WO2025089217A1 - COMPOUND FOR USE AS SUBSTRATE FOR EVALUATING ACTIVITY OF γ-D-GLUTAMYL-L-LYSYL ENDOPEPTIDASE - Google Patents

Abstract

[Problem] To provide a novel compound for use as a substrate for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase and a method for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase using the compound. [Solution] The present invention relates to a compound represented by formula (1) or a stereoisomer thereof, which is useful as a substrate for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase. [Formula 1] (In formula (1), A represents formula (2) or an amino-protecting group, and Z represents a detection group which becomes detectable upon liberation.) [Formula 2] (In formula (2), B represents a hydrogen atom, an optionally substituted N-acetyl-muramoyl group, or an acyl- or amino-protecting group.)

Description

Compounds to be used as substrates for evaluating the activity of gamma-D-glutamyl-L-lysyl endopeptidase

The present invention relates to a compound used as a substrate for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase, and a method for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase using the compound.

Lactic acid bacteria are known to have various functions, such as immune activation and anti-inflammatory effects, and contribute to health, and cell wall polysaccharides are thought to play an important role in immune activation.

In this way, structural analysis of cell wall polysaccharides has become important in elucidating the mechanism of action of the functionality of lactic acid bacteria. Traditionally, hot water extracts of cell walls have been used for structural analysis of cell wall polysaccharides. However, it was unclear whether hot water extracts maintained the original structure of cell wall polysaccharides.

Therefore, a method of structural analysis of cell wall polysaccharides using cell wall-lytic enzymes has attracted attention. Because enzymatic treatment can be carried out without using high heat, it is expected that cell wall polysaccharides can be analyzed in their original structure.

International Publication No. 2007/045192 International Publication No. WO 2019/036433

Schleifer, K. H., and Kandler, O., “Bacteriological Reviews”, American Society for Microbiology, 1972, p. 407-477. Regulski, K., et al., J Biol Chem, 2013, 288, p.20416-20426 Munneke, S., et al., Carbohydrate Research, 2015, vol. 414, p.1-7 C. Gallo-Rodriguez et al., Carbohydrate Research, 1998, vol. 305, p.163-170 A. Babic, S. Pecar, Tetrahedron: Asymmetry, 2008, Vol. 19, p.2265-2271 A. Babic, S. Pecar, Tetrahedron Letters, 2007, Vol. 48, p. 4403-4405 Imoto M., et al., Bull. Chem. Soc. Jpn., 1986, 59, 3207-3212 A. Nocentini et al., Bioorg. Med. Chem., 2015, vol. 23, p.6955-6966 A. Srinivas, et al., Chem. Med. Chem., 2017, vol. 12, p.1578-1584 Wu, X., et al., Beilstein J. Org. Chem., 2011, vol. 7, p.1030-1035 Herve Aloysius and Longqin Hu, Chem Biol Drug Des, 2015, vol. 86, p.837-843 J. Tsuji, “Palladium Reagent and Catalysts”, Wiley, 2004, Chap. 4, p.431-517

The present inventors focused on γ-D-glutamyl-L-lysyl endopeptidase (Lc-Lys2), one of the cell wall lytic enzymes of Lacticaceobacillus, for use in structural analysis of cell wall polysaccharides. A possible method for obtaining active γ-D-glutamyl-L-lysyl endopeptidase would be to clone γ-D-glutamyl-L-lysyl endopeptidase from Lacticaceobacillus, express it in Escherichia coli, and purify it. However, a method for accurately evaluating the activity of the produced γ-D-glutamyl-L-lysyl endopeptidase was unknown.

The present invention therefore aims to provide a novel compound that can be used as a substrate for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase, and a method for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase using the compound.

In order to solve the above problems, the present inventors searched for compounds that can be used to evaluate the activity of γ-D-glutamyl-L-lysyl endopeptidase (hereinafter also referred to as "Lc-Lys2"). A schematic diagram of the cell wall of the genus Lacticaceae is shown in Figure 1 (Non-Patent Documents 1-2). It was speculated that Lc-Lys2 cleaves between D-Glu or D-Gln and L-Lys in the cell wall peptide portion.

The present inventors synthesized synthetic substrates by introducing the fluorescent substance 7-amino-4-methylcoumarin (AMC) or the chromogenic substance 4-nitroaniline (4NA) to the terminus of the cleavage site by Lc-Lys2 as a substrate for evaluating the activity of the Lc-Lys2 enzyme, and binding a MurNAc-L-Ala derivative to enhance the specificity with the enzyme. Specifically, D-isoGln-MCA (MCA: 4-methylcoumarin-7-amide) and D-isoGln-pNA (pNA: p-nitroanilide) derivatives were synthesized.

The specificity of the synthetic substrates for Lc-Lys2 was evaluated by measuring the AMC or 4NA released by reaction of these synthetic substrates with Lc-Lys2 using a fluorometer or ultraviolet-visible spectrophotometer. Based on these findings, the inventors have discovered new compounds and methods of evaluating the activity of Lc-Lys2 that can be used to evaluate the activity of Lc-Lys2, and have completed the present invention.

The present invention provides a compound or a stereoisomer thereof represented by the following formula (1):

　　　　　　　・・・（１）
（上記式（１）中、Aは下記式（２）またはアミノ基の保護基を表し、Zは遊離すると検出可能となる検出基を表す。）

...(1)
(In the above formula (1), A represents the following formula (2) or a protecting group for an amino group, and Z represents a detection group that becomes detectable when released.)

　　　　　　・・・（２）
（上記式（２）中、Bは水素原子、置換基を有してもよいN-アセチル-ムラモイル基、アシル基またはアミノ基の保護基を表す。）
　本発明はまた、上記式（２）中のBによって表されるアミノ基の保護基がアルコキシカルボニル基、アルケニルオキシカルボニル基またはアラルキルオキシカルボニル基である、化合物またはその立体異性体を提供する。

... (2)
(In the above formula (2), B represents a hydrogen atom, an N-acetyl-muramoyl group which may have a substituent, an acyl group, or a protecting group for an amino group.)
The present invention also provides a compound or a stereoisomer thereof, wherein the protecting group for an amino group represented by B in the above formula (2) is an alkoxycarbonyl group, an alkenyloxycarbonyl group or an aralkyloxycarbonyl group.

The present invention also provides a compound or a stereoisomer thereof, in which the protecting group for the amino group represented by B in the above formula (2) is an Fmoc group (9-fluorenylmethyloxycarbonyl group), a Cbz group (benzyloxycarbonyl group), a Boc group (tert-butoxycarbonyl group) or an Alloc group (allyloxycarbonyl group).

The present invention also provides a compound or a stereoisomer thereof, in which the N-acetyl-muramoyl group, which may have a substituent and is represented by B in the above formula (2), is an N-acetyl-1-benzylmuramoyl group or an N-acetyl-1-benzyl-4,6-benzylidenemuramoyl group.

The present invention also provides a compound or a stereoisomer thereof, in which the acyl group represented by B in the above formula (2) is an acetyl group or a benzoyl group.

The present invention also provides a compound or a stereoisomer thereof, in which the protecting group for the amino group represented by A in the above formula (1) is an alkoxycarbonyl group, an alkenyloxycarbonyl group, or an aralkyloxycarbonyl group.

The present invention also provides a compound or a stereoisomer thereof, in which the protecting group for the amino group represented by A in the above formula (1) is an Fmoc group (9-fluorenylmethyloxycarbonyl group), a Cbz group (benzyloxycarbonyl group), a Boc group (tert-butoxycarbonyl group) or an Alloc group (allyloxycarbonyl group).

The present invention also provides a compound or a stereoisomer thereof, in which A in the above formula (1) is the following formula (3), (4), (5), (6), (7), (8) or (9).

　　　　　　　　　　　　　　　・・・（３）
（上記式（３）中、Acはアセチル基を表し、Bnはベンジル基を表す）

...(3)
(In the above formula (3), Ac represents an acetyl group, and Bn represents a benzyl group.)

　　　　　　　　　　　　　　・・・（４）
（上記式（４）中、Acはアセチル基を表し、Bnはベンジル基を表し、Phはフェニル基を表す）

...(4)
(In the above formula (4), Ac represents an acetyl group, Bn represents a benzyl group, and Ph represents a phenyl group.)

　　　　　　　　・・・（５）

...(5)

　　　　　　　　　　　　　　　・・・（６）

...(6)

　　　　　　　　　・・・（７）

...(7)

　　　　　　　　　　　　・・・（８）

...(8)

　　　　　　　　　　　　・・・（９）

...(9)

The present invention also provides a compound or a stereoisomer thereof in which Z in the above formula (1) represents a chromophore that emits color when released or a fluorescent group that emits fluorescence when released.

The present invention also provides a compound or a stereoisomer thereof, in which Z in the above formula (1) is the following formula (10), (11), (12), (13), (14) or (15).

　　　　　　　　　　　・・・（１０）

...(10)

　　　　　　　　　　　　　・・・（１１）

...(11)

　　　　　　　　　　　・・・（１２）

...(12)

　　　　　　　　　　　　　　・・・（１３）

...(13)

　　　　　　　　　　　　　　　　・・・（１４）

...(14)

　　　　　　　　　・・・（１５）
 

...(15)

The present invention also provides the above compound or a stereoisomer thereof for use as a substrate for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase.

The present invention also provides a method for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase, comprising the steps of reacting γ-D-glutamyl-L-lysyl endopeptidase with a compound represented by the following formula (1) or a stereoisomer thereof, and detecting the liberated Z.

　　　　　　・・・（１）
（上記式（１）中、Aは下記式（２）またはアミノ基の保護基を表し、Zは遊離すると検出可能となる検出基を表す。）

...(1)
(In the above formula (1), A represents the following formula (2) or a protecting group for an amino group, and Z represents a detection group that becomes detectable when released.)

　　　　　　・・・（２）
（上記式（２）中、Bは水素原子、置換基を有してもよいN-アセチル-ムラモイル基、アシル基またはアミノ基の保護基を表す。）

... (2)
(In the above formula (2), B represents a hydrogen atom, an N-acetyl-muramoyl group which may have a substituent, an acyl group, or a protecting group for an amino group.)

The present invention can provide a novel compound that can be used as a substrate for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase. The present invention can also provide a method for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase using the compound. Therefore, the present invention can be used to select γ-D-glutamyl-L-lysyl endopeptidase that has activity useful for structural analysis of cell wall polysaccharides of lactic acid bacteria.

1 shows a schematic diagram of the cell wall of Lacticaceobacter sp.

Terms used in this specification are defined below.

　"Alkyl group" refers to a linear or branched alkyl group having 1 to 20 carbon atoms. Examples of alkyl groups include linear or branched lower alkyl groups having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, and 2-ethylbutyl, and linear or branched alkyl groups having 7 to 20 carbon atoms, such as heptyl, octyl, nonyl, and decyl. The alkyl group may have a ring formed as a part thereof.

"Alkoxy group" refers to an alkoxy group having the above alkyl group. Examples of alkoxy groups include methyloxy (methoxy), ethyloxy (ethoxy), n-propyloxy (n-propoxy), isopropoxy, n-butyloxy (n-butoxy), isobutoxy, s-butoxy, tert-butoxy, and n-pentyloxy (n-pentoxy).

The term "acyl group" includes aliphatic acyl groups and aromatic acyl groups. Examples of aliphatic acyl groups include formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl, 14-methylpentadecanoyl, 13,13-dimethyltetradecanoyl, heptadecanoyl, 15-methylpentadecanoyl, 20-methylpentadecanoyl, 25-methylpentadecanoyl, 26-methylpentadecanoyl, 27-methylpentadecanoyl, 28-methylpentadecanoyl, 29-methylpentadecanoyl, 30-methylpentadecanoyl, 31-methylpentadecanoyl, 32-methylpentadecanoyl, 33-methylpentadecanoyl, 34-methylpentadecanoyl, 35-methylpentadecanoyl, 36-methylpentadecanoyl, 37-methylpentadecanoyl, 38-methylpentadecanoyl, 39-methylpentadecanoyl, 40-methylpentadecanoyl, 41-methylpentadecanoyl, 42-methylpentadecanoyl, 43-methylpentadecanoyl, 44-methylpentadecanoyl, 45-methylpentadecanoyl, 46-methylpentadecanoyl, 47-methylpentadecanoyl, 48-methylpentadecanoyl, 49-methylpentadecanoyl, 50-methylpentadecanoyl, -alkylcarbonyl groups such as methylhexadecanoyl, octadecanoyl, 1-methylheptadecanoyl, nonadecanoyl, eicosanoyl and henaicosanoyl; carboxylated alkylcarbonyl groups such as succinoyl, glutaroyl and adipoyl; halogeno lower alkylcarbonyl groups such as chloroacetyl, dichloroacetyl, trichloroacetyl and trifluoroacetyl; lower alkoxy lower alkylcarbonyl groups such as methoxyacetyl; and unsaturated alkylcarbonyl groups such as (E)-2-methyl-2-butenoyl. Aromatic acyl groups include, for example, arylcarbonyl groups such as benzoyl, α-naphthoyl, and β-naphthoyl; halogenoarylcarbonyl groups such as 2-bromobenzoyl and 4-chlorobenzoyl; lower alkylated arylcarbonyl groups such as 2,4,6-trimethylbenzoyl and 4-toluoyl; lower alkoxylated arylcarbonyl groups such as 4-anisoyl; carboxylated arylcarbonyl groups such as 2-carboxybenzoyl, 3-carboxybenzoyl, and 4-carboxybenzoyl; nitrated arylcarbonyl groups such as 4-nitrobenzoyl and 2-nitrobenzoyl; lower alkoxycarbonylated arylcarbonyl groups such as 2-(methoxycarbonyl)benzoyl; and arylated arylcarbonyl groups such as 4-phenylbenzoyl.

　"Aralkyl group" refers to an alkyl group having 1 to 6 carbon atoms substituted with an aryl group. Examples of aralkyl groups include benzyl, α-naphthylmethyl, β-naphthylmethyl, indenylmethyl, phenanthrenylmethyl, anthracenylmethyl, diphenylmethyl, triphenylmethyl, α-naphthyldiphenylmethyl, 9-anthrylmethyl, 4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4-cyanobenzyl, 1-phenethyl, 2-phenethyl, 1-naphthylethyl, 2-naphthylethyl, 1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, 1-naphthylpropyl, 2 ... These include propyl, 3-naphthylpropyl, 1-phenylbutyl, 2-phenylbutyl, 3-phenylbutyl, 4-phenylbutyl, 1-naphthylbutyl, 2-naphthylbutyl, 3-naphthylbutyl, 4-naphthylbutyl, 1-phenylpentyl, 2-phenylpentyl, 3-phenylpentyl, 4-phenylpentyl, 5-phenylpentyl, 1-naphthylpentyl, 2-naphthylpentyl, 3-naphthylpentyl, 4-naphthylpentyl, 5-naphthylpentyl, 1-phenylhexyl, 2-phenylhexyl, 3-phenylhexyl, 4-phenylhexyl, 5-phenylhexyl, 6-phenylhexyl, 1-naphthylpentyl, 2-naphthylpentyl, 3-naphthylpentyl, 4-naphthylpentyl, 5-naphthylpentyl and 6-naphthylpentyl.

[Compound]
The present invention provides a compound represented by the following formula (1) or a stereoisomer thereof:

　　　　　　・・・（１）

...(1)

In the above formula (1), A represents the following formula (2) or a protecting group for an amino group.

　　　　　　・・・（２）

... (2)

In the above formula (2), B represents a hydrogen atom, an N-acetyl-muramoyl group which may have a substituent, an acyl group, or a protecting group for an amino group.

When B in the above formula (2) represents an N-acetyl-muramoyl group which may have a substituent, N-acetylmuramic acid (MurNAc) which may have a substituent is bonded to B in formula (2) through an amide bond. The N-acetyl-muramoyl group represented by B may have one or more substituents, or may not have a substituent. The substituent may be, for example, an alkyl group having 1 to 6 carbon atoms, an aralkyl group having 7 to 10 carbon atoms, or a benzylidene group. The substituent may also be an alcohol protecting group, for example, a benzyl group (Bn), a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, a p-methoxybenzyl group, or a benzylidene group. The N-acetyl-muramoyl group represented by B may have, for example, the 4-OH group and the 6-OH group protected by the benzylidene carbon. B is preferably an N-acetyl-1-benzylmuramoyl group or an N-acetyl-1-benzyl-4,6-benzylidenemuramoyl group.

The acyl group represented by B in the above formula (2) can be, for example, an aliphatic acyl group having 1 to 10 carbon atoms and an aromatic acyl group. The acyl group represented by B is preferably an acetyl group or a benzoyl group.

The amino protecting group represented by B in the above formula (2) is not particularly limited, and may be an amide protecting group, a phthalimide protecting group, a carbamate protecting group, or a sulfonamide protecting group. The amino protecting group represented by B is preferably a carbamate protecting group, such as an alkoxycarbonyl group, an alkenyloxycarbonyl group, or an aralkyloxycarbonyl group. Examples of carbamate protecting groups include the Fmoc group (9-fluorenylmethyloxycarbonyl group), the Cbz group (benzyloxycarbonyl group), the Boc group (tert-butoxycarbonyl group), the Alloc group (allyloxycarbonyl group), the Aoc group (tert-amyloxycarbonyl group), and the Troc group (2,2,2-triethoxycarbonyl group). Examples of amide protecting groups include the formyl group, the acetyl group (Ac), and the trifluoroacetyl group (TFA). Phthalimide-type protecting groups include, for example, a phthaloyl group (Phth) and the like. Sulfonamide-type protecting groups include, for example, a 3-nitro-2-pyridinesulfenyl group (Npys), a 2-nitrobenzenesulfonyl group (Ns) and a (2-trimethylsilyl)-ethanesulfonyl group (SES) and the like. The protecting group for the amino group represented by B is preferably an alkoxycarbonyl group, an alkenyloxycarbonyl group or an aralkyloxycarbonyl group, more preferably an Fmoc group, a Cbz group, a Boc group or an Alloc group, and even more preferably an Fmoc group.

In the above formula (1), the protecting group of the amino group represented by A is not particularly limited, and an amide type protecting group, a phthalimide type protecting group, a carbamate type protecting group, a sulfonamide type protecting group, etc. can be used. The protecting group of the amino group represented by A can be preferably a carbamate type protecting group, such as an alkoxycarbonyl group, an alkenyloxycarbonyl group, and an aralkyloxycarbonyl group. Examples of the carbamate type protecting group include, for example, the Fmoc group (9-fluorenylmethyloxycarbonyl group), the Cbz group (benzyloxycarbonyl group), the Boc group (tert-butoxycarbonyl group), the Alloc group (allyloxycarbonyl group), the Aoc group (tert-amyloxycarbonyl group), and the Troc group (2,2,2-triethoxycarbonyl group). Examples of the amide type protecting group include, for example, a formyl group, an acetyl group (Ac), and a trifluoroacetyl group (TFA). Phthalimide-type protecting groups include, for example, a phthaloyl group (Phth) and the like. Sulfonamide-type protecting groups include, for example, a 3-nitro-2-pyridinesulfenyl group (Npys), a 2-nitrobenzenesulfonyl group (Ns) and a (2-trimethylsilyl)-ethanesulfonyl group (SES) and the like. The protecting group for the amino group represented by A is preferably an alkoxycarbonyl group, an alkenyloxycarbonyl group or an aralkyloxycarbonyl group, more preferably an Fmoc group, a Cbz group, a Boc group or an Alloc group, and even more preferably an Fmoc group.

A in the above formula (1) can be, for example, the following formula (3), (4), (5), (6), (7), (8) or (9).

　　　　　　　　　　　　・・・（３）
（上記式（３）中、Acはアセチル基を表し、Bnはベンジル基を表す）

...(3)
(In the above formula (3), Ac represents an acetyl group, and Bn represents a benzyl group.)

　　　　　　　　　　　　　・・・（４）
（上記式（４）中、Acはアセチル基を表し、Bnはベンジル基を表し、Phはフェニル基を表す）

...(4)
(In the above formula (4), Ac represents an acetyl group, Bn represents a benzyl group, and Ph represents a phenyl group.)

　　　　　　　　・・・（５）

...(5)

　　　　　　　　　　　　　　　・・・（６）

...(6)

　　　　　　　　　　・・・（７）

...(7)

　　　　　　　　　　　・・・（８）

...(8)

　　　　　　　　　　　　・・・（９）
 

...(9)

In the above formula (1), Z represents a detection group that becomes detectable when released. Z can be, for example, a chromophore that develops color when released or a fluorescent group that emits fluorescence when released. Z can be, for example, a detection group that is colorless or non-fluorescent before cleavage by the enzyme, but becomes colored or fluorescent when released following cleavage by the enzyme.

In the present invention, the chromophore represented by Z is not particularly limited as long as it is a compound that emits color when released, but can be, for example, a paranitroaniline derivative. In the present invention, the fluorescent group represented by Z is not particularly limited as long as it is a compound that emits fluorescence when released, but can be, for example, a coumarin derivative and a fluorescein derivative. Z is preferably the following formula (10), (11), (12), (13), (14) or (15), and particularly preferably (10), (11) or (12).

　　　　　　　　　　　・・・（１０）

...(10)

　　　　　　　　　　　・・・（１１）

...(11)

　　　　　　　　　　・・・（１２）

...(12)

　　　　　　　　　　　　　　　・・・（１３）

...(13)

　　　　　　　　　　　　　　　・・・（１４）

...(14)

　　　　　　　　　・・・（１５）
 

...(15)

Z can be, for example, methylcoumarinamide (MCA) represented by the above formula (10) or paranitroanilide (pNA) represented by the above formula (12).

When aminomethylcoumarin (AMC) is released from a substrate containing MCA by an enzyme reaction, it emits strong fluorescence, and the enzyme reaction can be detected by detecting this change in fluorescence using a fluorometer or other device.

When 4-nitroaniline is released from a substrate containing pNA by an enzyme reaction, it turns yellow, and the enzyme reaction can be detected by detecting this color change using a UV-visible spectrophotometer or similar.

As used herein, "stereoisomers" refer to compounds that have the same chemical structure but differ in the arrangement of atoms or groups in space. When multiple stereoisomers exist, the compound of the present invention may be a mixture of two or more stereoisomers.

The compound of the present invention can preferably be benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (compound 63B) or benzyl-α-MurNAc-L-Ala-D-isoGln-pNA (compound 64B).

[Substrates for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase]
The compound of the present invention can be used as a substrate for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase. The γ-D-glutamyl-L-lysyl endopeptidase can be, but is not limited to, γ-D-glutamyl-L-lysyl endopeptidase (Lc-Lys2) of lactic acid bacteria, particularly of the genus Lacticaceibacillus. The genus Lacticaceibacillus includes, for example, Lacticaceibacillus casei, Lacticaceibacillus paracasei, and Lacticaceibacillus rhamnosus.

Lc-Lys2 is one of the cell wall lytic enzymes of the Lacticaceobacterium genus. Lc-Lys2 has the activity of cleaving the D-isoGln site of the peptide portion of the cell wall. The compound of the present invention contains Z, which is released and detectable when cleaved by Lc-Lys2, so the activity of Lc-Lys2 can be evaluated by detecting this Z.

[Method for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase]
The present invention also provides a method for assessing the activity of γ-D-glutamyl-L-lysyl endopeptidase.

In the present invention, the "activity" of γ-D-glutamyl-L-lysyl endopeptidase means the activity of cleaving the D-isoGln site in the cell wall peptide portion of Lacticaceobacter sp.

In the present invention, "assessing activity" includes not only assessing the presence or absence of activity to cleave a substrate, but also assessing the strength of the activity to cleave a substrate. In the present invention, "activity" can be expressed, for example, by the rate at which the substrate is cleaved, the amount and proportion of products (liberated substances) generated by cleavage of the substrate, the time it takes for the substrate to be cleaved to a specified proportion, etc.

The method of the present invention includes a step of reacting γ-D-glutamyl-L-lysyl endopeptidase with the compound of the present invention or its stereoisomer, and a step of detecting the released Z. In the method of the present invention, the compound or its stereoisomer is used as a substrate for γ-D-glutamyl-L-lysyl endopeptidase.

In this specification, the expression "reacts" between an enzyme and a substrate means that the enzyme catalyzes the conversion of the substrate into one or more different products. In the present invention, the expression "reacts" between γ-D-glutamyl-L-lysyl endopeptidase and a compound of the present invention means that γ-D-glutamyl-L-lysyl endopeptidase liberates the detection group represented by Z in the above formula (1) from the compound of the present invention. The reaction can be caused by contacting the enzyme with the substrate.

In the present invention, "detecting" Z includes detecting the presence or absence of free Z, and detecting changes such as increases and decreases in the amount and/or proportion of free Z.

The γ-D-glutamyl-L-lysyl endopeptidase used in the method of the present invention may be an enzyme purified from Escherichia coli or lactic acid bacteria, or may be an extract from bacteria that may contain γ-D-glutamyl-L-lysyl endopeptidase. Furthermore, the γ-D-glutamyl-L-lysyl endopeptidase used in the method of the present invention does not necessarily have to be an active enzyme, and may be a candidate substance whose function as γ-D-glutamyl-L-lysyl endopeptidase is unknown.

In the step of reacting γ-D-glutamyl-L-lysyl endopeptidase with the above-mentioned compound or its stereoisomer, the reaction can be carried out, for example, by contacting γ-D-glutamyl-L-lysyl endopeptidase with the above-mentioned compound in a reaction solution for a certain period of time. The reaction temperature is not particularly limited, but can be, for example, 20 to 50°C, preferably 30 to 40°C, and more preferably 37°C. The reaction time is not particularly limited, and can be appropriately set between 5 minutes and 24 hours, for example.

In this specification, "contacting" an enzyme with a substrate means bringing the enzyme and substrate close enough to each other to perform catalytic reactions. "Contacting" an enzyme with a substrate includes, for example, mixing the enzyme with the substrate and adding the enzyme to the substrate. For example, the enzyme and substrate can be brought into contact by adding them to a reaction solution and mixing them by stirring or the like.

The reaction solution is not particularly limited, but for example, phosphate buffer and acetate buffer can be used.

In the process of detecting released Z, depending on the type of Z, the presence, absence, amount and/or proportion of Z can be measured using an instrument capable of detecting Z. If Z is a chromophore that emits color upon release, the presence, absence, amount and/or proportion of released Z can be measured using an ultraviolet-visible spectrophotometer or the like. If Z is a fluorescent group that emits fluorescence upon release, the presence, absence, amount and/or proportion of released Z can be measured using a fluorometer or the like.

In the method of the present invention, the γ-D-glutamyl-L-lysyl endopeptidase used may be evaluated as having activity if the presence of Z is detected in the step of detecting released Z. Alternatively, the theoretical amount of Z produced may be calculated in advance, and the γ-D-glutamyl-L-lysyl endopeptidase used may be evaluated as having activity if the ratio (%) of the amount of Z detected to this theoretical amount of production exceeds a preset threshold. The threshold is not particularly limited, but may be set to any value, such as 6%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%. In the method of the present invention, the strength of activity of the γ-D-glutamyl-L-lysyl endopeptidase used may be evaluated based on the amount and/or ratio of released Z.

[Production of Compounds]
The compound of the present invention or its stereoisomer can be produced by the following methods, the methods described in the Examples, or methods modified therefrom. The reaction conditions can be optimized as appropriate depending on the compound and solvent used.

(Scheme 1: Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-L,D-Ala-OH (Compounds 7 and 8))
Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-OH (compound 7) and benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-OH (compound 8) can be prepared according to Scheme 1 below.

Benzyl 2-acetamido-2-deoxy-α-D-glucopyranoside (compound 2) can be obtained by boiling N-acetyl-D-glucosamine (compound 1) with benzyl alcohol (BnOH) and p-toluenesulfonic acid monohydrate (p-TSA·H ₂ O) in toluene under reflux (see Non-Patent Document 3).

Next, compound 2 is heated with benzaldehyde dimethyl acetal and p-TSA·H ₂ O in N,N-dimethylformamide (DMF) and dehydrated, and the hydroxyl groups at the 4 and 6 positions are benzylidene-protected to give benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α-D-glucopyranoside (compound 3) (see non-patent documents 4-6).

Then, (S)-(-)-2-chloropropionic acid is introduced into the 3-position hydroxyl group of compound 3 using NaH as a base in DMF solvent under an argon atmosphere to synthesize 2-(2-(R)-(2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionic acid (benzyl-4,6-O-benzylidene-α-MurNAc) (compound 4) (see Patent Document 1 and Non-Patent Documents 4 to 7).

　Protected MurNAc (compound 4) was condensed with L-alanine methyl ester hydrochloride or D-alanine methyl ester hydrochloride in the presence of N-methylmorpholine in DMF using N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) to give (S)-methyl 2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionate. (R)-methyl 2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyrano-syl)oxy)propionamido)-propionate (benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-OMe) (compound 6) can be synthesized (see non-patent literature 4 to 6).

Compound 5 and compound 6 can be hydrolyzed with 0.5 mol/L KOH in MeOH solvent to obtain (S)-2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionic acid (benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-OH) (compound 7) and (R)-2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-gluco-pyranosyl)oxy)propionamido)propionic acid (benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-OH) (compound 8).

(Scheme 2: Synthesis of D-Glu-MCA, D-Gln-MCA, D-isoGln-MCA and D-isoGln-pNA derivatives)
D-Glu-MCA, D-Gln-MCA, D-isoGln-MCA and D-isoGln-pNA derivatives can be synthesized according to Scheme 2 below.

Sodium hydroselenide (NaSeH) can be prepared from selenium (powder; Se) and sodium borohydride (NaBH ₄ ) in isopropanol (IPA) immediately before use. 7-Azido-4-methylcoumarin (compound 10) (see Non-Patent Documents 8-9) and 1-azido-4-nitrobenzene (compound 22) can be prepared from AMC and 4-nitroaniline hydrochloride.

tert-Butoxycarbonyl (Boc)-D-Glu(OBn)-OH (compound 9), 9-fluorenylmethoxycarbonyl (Fmoc)-D-Glu(OAll)-OH (compound 12), Fmoc-D-Glu(OH)-OAll (compound 14), Boc-D-Gln-OH (compound 16), Fmoc-D-Gln-OH (compound 18) and Fmoc-D-isoGln-OH (compound 20) were reacted with isopropyl chloroformate (toluene solution) in the presence of N-methylpiperidine in THF to give mixed acid anhydrides, which were then reacted with freshly prepared sodium hydroselenide (NaSeH) to give selenium. Through the nido intermediate, it is possible to synthesize Boc-D-Glu(OBn)-MCA (compound 11), Fmoc-D-Glu(OAll)-MCA (compound 13), Fmoc-D-Glu(MCA)-OAll (compound 15), Boc-D-Gln-MCA (compound 17), Fmoc-D-Gln-MCA (compound 19), Fmoc-D-isoGln-MCA (compound 21), and Fmoc-D-isoGln-pNA (compound 23) by reacting with 7-azido-4-methylcoumarin (compound 10) and 1-azido-4-nitrobenzene (compound 22) (see non-patent literature 10-11).

The Boc-protected form (compound 11) can be treated with trifluoroacetic acid (TFA) _{in CH2Cl2} to give the Boc-deprotected form HD-Glu(OBn)-MCA·TFA (compound 24), which can then be treated further with aqueous _NaHCO3 to give the free base (compound 25).

The Boc-protected form (compound 17) can be reacted in TFA/CH ₂ Cl ₂ /H ₂ O at room temperature and treated with Amberlyst® A-26 to give HD-Gln-MCA (compound 26) as a free base.

Fmoc-protected forms (compounds 13, 15, 19, 21, 23) can also be treated with 20% piperidine/DMF to give the free bases H-D-Glu(OAll)-MCA (compound 28) (All; allyl), H-D-Glu(MCA)-OAll (compound 27), H-D-Gln-MCA (compound 26), H-D-isoGln-MCA (compound 30) and H-D-isoGln-pNA (compound 31). When compound 13 is de-Fmoc-converted, hydrolysis of the allyl ester occurs together with the free base (compound 28) to give H-D-Glu(OH)-MCA (compound 29).

(Scheme 3: Synthesis of HL-Ala-D-Glu-MCA, HL-Ala-D-Gln-MCA, HL-Ala-D-isoGln-MCA and HL-Ala-D-isoGln-pNA)
HL-Ala-D-Glu-MCA, HL-Ala-D-Gln-MCA, HL-Ala-D-isoGln-MCA and HL-Ala-D-isoGln-pNA can be synthesized according to Scheme 3 below.

Boc-L-Ala-OH (compound 32) is reacted with isopropyl chloroformate in the presence of N-methylpiperidine in THF to give a mixed acid anhydride, which is then reacted with H-D-Glu(OBn)-MCA (compound 25) and H-D-Gln-MCA (compound 26) to give Boc-L-Ala-D-Glu(OBn)-MCA (compound 35) and Boc-L-Ala-D-Gln-MCA (compound 36).

In addition, Fmoc-L-Ala-OH hydrate (compound 33) was condensed with HD-Glu(MCA)-OAll (compound 27), HD-Gln-MCA (compound 26), HD-isoGln-MCA (compound 30), and HD-isoGln-pNA (compound 31) in the presence of 1-hydroxybenzotriazole monohydrate (HOBt·H ₂ O) in DMF using N,N-diisopropylethylamine (DIEA) as a base and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) as a condensation agent to give Fmoc-L-Ala-D-Glu(MCA)-OAll (compound 37), Fmoc-L-Ala-D-Gln-MCA (compound 38), Fmoc-L-Ala-D- isoGln-MCA (compound 39) and Fmoc-L-Ala-D-isoGln-pNA (compound 40) can be obtained.

The Boc-protected form (compound 35) can be de-Bocated using TFA in _CH2Cl2 to give HL-Ala-D-Glu(OBn)-MCA·TFA (compound 41). The _Fmoc -protected forms (compounds 37, 38, 39, and 40) can be treated with 20% piperidine/DMF to give HL-Ala-D-Glu(MCA)-OAll (compound 42), HL-Ala-D-Gln-MCA (compound 44), HL-Ala-D-isoGln-MCA (compound 45), and HL-Ala-D-isoGln-pNA (compound 46).

When compound 37 is de-Fmoc-conjugated, hydrolysis of the allyl ester occurs, and H-L-Ala-D-Glu(MCA)-OH (compound 43) can be obtained as the main product. Compound 45 can also be treated with 5-10% HCl/MeOH to convert it to its hydrochloride salt.

HL-Ala-D-isoGln-pNA (compound 46) can be treated with acetyl chloride and benzoyl chloride in the presence of triethylamine (TEA) _{in CH2Cl2} to give Ac-L-Ala-D-isoGln-pNA (compound 47) and Bz-L-Ala-D-isoGln-pNA (compound 48).

(Scheme 4: Synthesis of D-Glu(OH)-MCA derivative (debenzylation))
The D-Glu(OH)-MCA derivative can be synthesized by debenzylation according to Scheme 4 below.

Boc-D-Glu(OBn)-MCA (compound 11), Boc-L-Ala-D-Glu(OBn)-MCA (compound 35), H-L-Ala-D-Glu(OBn)-MCA·TFA (compound 41) and H-D-Glu(OBn)-MCA·TFA (compound 24) can be hydrogenolyzed in EtOH under a hydrogen atmosphere using 5% Pd/C as a catalyst to obtain the debenzylated forms Boc-D-Glu(OH)-MCA (compound 50), Boc-L-Ala-D-Glu(OH)-MCA (compound 51), H-L-Ala-D-Glu(OH)-MCA·TFA (compound 52) and H-D-Glu(OH)-MCA·TFA (compound 53).

Scheme 5: Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-L, D-Ala-D-Glu-MCA, -D-Gln-MCA, -D-isoGln-MCA and -D-isoGln-pNA derivatives.
Benzyl-4,6-O-benzylidene-α-MurNAc-L, D-Ala-D-Glu-MCA, -D-Gln-MCA, -D-isoGln-MCA and -D-isoGln-pNA derivatives can be synthesized according to Scheme 5 below.

 
 

 
 

Protected MurNAc-L-Ala-D-Glu-MCA, protected MurNAc-L-Ala-D-Gln-MCA, protected MurNAc-L-Ala-D-isoGln-MCA and protected MurNAc-L-Ala-D-isoGln-pNA derivatives can be synthesized by two synthetic routes.

Benzyl-4,6-O-benzylidene-α-MurNAc (compound 4) was condensed with H-L-Ala-D-Glu(MCA)-OAll (compound 42), H-L-Ala-D-Gln-MCA (compound 44) and H-L-Ala-D-isoGln-pNA (compound 46) in DMF solvent using N-methylmorpholine as a base and HBTU as a condensing agent (method a), to give the vector. It is possible to synthesize benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(MCA)-OAll (compound 54), benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Gln-MCA (compound 55), and benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-pNA (compound 56).

In addition, benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-OH (compound 7) was reacted with HD-Glu(OAll)-MCA (compound 28), HD-Glu(MCA)-OAll (compound 27), HD-isoGln-MCA (compound 30), and HD-isoGln-pNA (compound 31) in DMF with HOBt·H ₂ Condensation using DIEA as a base and EDCI as a condensing agent in the presence of O (Method b) can give benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(OAll)-MCA (Compound 57), benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(MCA)-OAll (Compound 54), and benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-MCA (Compound 58).

Benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-OH (compound 8) can be condensed with compound 31 by method b to obtain benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-D-isoGln-pNA (compound 59). In addition, benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-pNA (compound 56) can be obtained by condensing compound 7 with compound 31 by method a.

Scheme 6: Synthesis of benzyl-α-MurNAc-Ala-D-Glu-MCA, -D-Gln-MCA, -D-isoGln-MCA and -D-isoGln-pNA derivatives.
The 4,6-benzylidene and allyl groups can be removed to synthesize benzyl-α-MurNAc-Ala-D-Glu-MCA, -D-Gln-MCA, -D-isoGln-MCA and -D-isoGln-pNA derivatives according to Scheme 6 below.

The benzyl-4,6-O-benzylidene-α-MurNAc derivatives (compounds 55, 57, 54, 58, 56 and 59) can be debenzylidene-substituted by heating in 75% AcOH to give benzyl-α-MurNAc-L-Ala-D-Gln-MCA (compound 60), benzyl-α-MurNAc-L-Ala-D-Glu(OAll)-MCA (compound 61), benzyl-α-MurNAc-L-Ala-D-Glu(MCA)-OAll (compound 62), benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (compound 63), benzyl-α-MurNAc-L-Ala-D-isoGln-pNA (compound 64) and benzyl-α-MurNAc-D-Ala-D-isoGln-pNA (compound 65).

In addition, the allyl-protected derivatives (compounds 61 and 62) can be de-allylated using Pd(OAc) ₂ - _PPh3 catalyst in the presence of AcOH and 4-methylmorpholine as a nucleophile in _CH3CN - _H2O (see Patent Document 2 and Non-Patent Document 12) to give benzyl-α-MurNAc-L-Ala-D-Glu(OH)-MCA (compound 66) and benzyl-α-MurNAc-L-Ala-D-Glu(MCA)-OH (compound 67).

In the following examples, the reagents used in the reactions were commercially available and were used without purification.

The following abbreviations are used in the specification and examples:
4NA 4-Nitroaniline
AcOEt Ethyl acetate
AcOH Acetic acid
AMC 7-amino-4-methylcoumarin
Boc tert-butoxycarbonyl
DIEA N,N-Diisopropylethylamine
DMF N,N-Dimethylformamide
DMSO Dimethyl sulfoxide
EDCI 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide
eq. Molar equivalent
EtOH Ethanol
Fmoc 9-Fluorenylmethoxycarbonyl
HBTU O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate
HOBt Hydroxybenzotriazole
IPA Isopropanol
IPE Isopropyl Ether
KOH Potassium hydroxide
LCMS Liquid Chromatography Mass Spectrometry
Ph Phenyl
pNA p-nitroanilide
p-TSA・H ₂ O p-Toluenesulfonic acid・Monohydrate
Mass
MeOH Methanol
NaSeH Sodium hydroselenide
n-Hex. Normal hexane
NMR Nuclear Magnetic Resonance
Pd/C Palladium Carbon
quant. Quantitative yield
THF Tetrahydrofuran
TFA Trifluoroacetic acid
TLC Thin Layer Chromatography

The progress of the synthesis reaction was monitored by thin layer chromatography (TLC) (Merck TLC Silica gel 60 F ₂₅₄ Glass plate). Anisaldehyde was used for color development. Silica gel column chromatography was performed using a flash automatic purification system (Biotage Isolera One), and the columns used were Biotage (registered trademark) SNAP Cartridge KP-Sil 10g, SNAP Ultra 10g (HP-Sphere ^TH 25μm), SNAP Ultra 25g (HP-Sphere ^TH 25μm), SNAP Ultra 50g (HP-Sphere ^TH 25μm), Sfar Silica HC D 25g (High Capacity Duo 20μm), Sfar Silica HC D 50g (High Capacity Duo 20μm), Sfar Silica HC D 100g (High Capacity Duo 20μm), Sfar Silica D 25g (Duo 60μm), or Shoko Science Co., Ltd. Purif-Pack (registered trademark)-EX SI-25μm, SIZE 200, as appropriate.

^1H nuclear magnetic resonance spectra were measured using a JEOL JNM-ECX-400P (400 MHz). Chemical shifts were measured with tetramethylsilane (0.00 ppm) as the internal standard in deuterated chloroform and deuterated dimethylsulfoxide. Liquid chromatography mass spectrometry (LCMS) spectra were measured using a Waters Acqity UPLC H-Class system [Column: Acquity UPLC BEH C18 61.7 μm 2.1*50 mm, Mobile phase: 0.1%HCO2H (A)/ _CH3CN (B), Flow rate 0.7 mL/min., Gradient 0 min. ₍ A: 91%) -3.2 min. (A: 19%) -3.7 min. (A: 19 > B: 91) -5 min. (B: 91)].

(Production of γ-D-glutamyl-L-lysyl endopeptidase)
The γ-D-glutamyl-L-lysyl endopeptidase used in this example was Lc-Lys2 contained in Lacticaseibacillus paracasei strain BL23.

The gene for the cell wall lytic enzyme Lc-Lys2 (γ-D-glutamyl-L-lysyl endopeptidase) derived from an endogenous phage was cloned from the Lacticaseibacillus paracasei BL23 strain, and the enzyme was expressed in E. coli by ligating it to an E. coli expression vector (Lc-Lys2). Lc-Lys2 was purified by dissolving the inclusion bodies of the insoluble fraction with a denaturant, and active enzyme was produced by normalizing the protein folding using the unfolding method.

Example 1: Synthesis of synthetic substrates
Each compound was synthesized by the method described below.

(Synthesis of benzyl 2-acetamido-2-deoxy-α-D-glucopyranoside (compound 2))

N-acetyl-D-glucosamine (compound 1) (25.0 g, 113 mmol), p-TSA·H ₂ O (1.9 g, 10 mmol) were suspended in toluene (300 mL) and benzyl alcohol (180 mL), and the suspension was boiled and refluxed for 3 hours with a Dean-Stark apparatus attached. The reaction mixture was cooled to room temperature, and NaHCO ₃ (1.26 g, 15 mmol) was dissolved in H ₂ O (15 mL) and added, and the toluene was distilled off under reduced pressure. The residue (black liquid) was cooled to room temperature, and AcOEt (120 mL) and n-Hex. (620 mL) were added and stirred. The precipitated light brown solid was collected by filtration, and the filtered product was suspended in a mixture of n-Hex. (240 mL)/AcOEt (120 mL) and washed with stirring.

The suspension was filtered, washed with n-Hex., and recrystallized from IPA (150 mL). The precipitate was filtered, washed with cold IPA (45 mL) and isopropyl ether (IPE; 50 mL x 2), and dried (50°C, reduced pressure) to obtain 15.17 g of crude compound as a light brown solid. The crude was dissolved in 20% MeOH/CHCl ₃ , silica gel (FL100D; 50 g) was added, and the mixture was concentrated to dryness under reduced pressure. The mixture was purified by silica gel column chromatography (5% to 20% MeOH/CHCl ₃ ) to obtain compound 2 (10.32 g, 29.3%) as a white to slightly brown solid.
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ ppm: 7.81 (d, 1H, J = 8.0 Hz, NH; D ₂ O exchange), 7.42-7.24 (m, 5H, Ph-H), 5.01 (d, 1H, J = 5.7 Hz; D ₂ O exchange), 4.72 (d, 1H, J =5.7 Hz; D ₂ O exchange), 4.70 (d, 1H, J = 3.7 Hz, C1-βH), 4.67 (d, 1H, Jgem = 12.6 Hz, Ph-CH ₂ a), 4.54 (t, 1H, J = 6.0 Hz, C6-OH; D ₂ O exchange), 4.42(d, 1H, Jgem = 12.6 Hz, Ph-CH ₂ b), 3.73-3.61 (m, 2H), 3.58-3.42 (m, 3H), 3.21-3.11 (m, 1H), 1.83 (s, 3H, NHCOCH ₃ ).
LCMS m/z: 312.1 [M+H] ⁺ . (Exact Mass: 311.14)
Purity (LCMS): 100.00%

(Synthesis of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α-D-glucopyranoside (compound 3))

Benzyl 2-acetamido-2-deoxy-α-D-glucopyranoside (compound 2) (10.32 g, 33.1 mmol) was dissolved in dehydrated DMF (80 mL), and benzaldehyde dimethyl acetal (6.9 mL, 46.5 mmol, 1.4 eq.) and p-TSA·H ₂ O (63 mg, 0.3 mmol, 0.01 eq.) were added. The mixture was placed on a rotary evaporator, and the solvent was gently removed under reduced pressure every hour (about 15 min), and heated at 60 °C for 5 h. The solvent was removed under reduced pressure to obtain a white solid residue. The residue was suspended in 2% NaHCO ₃ aqueous solution (50 mL), and the mass was crushed, collected by filtration, washed with water and IPE, and dried (60 °C, reduced pressure) to obtain compound 3 (13.07 g, 98.7%) as a white solid.
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 8.00 (d, 1H, J = 8.2 Hz, NH; D ₂ O exchange), 7.49-7.27 (m, 10H, Ph-H X 2), 5.62 (s, 1H, PhCH), 5.19 (d, 1H, J = 3.4 Hz, C3-OH; D ₂ O exchange), 4.79 (d, 1H, J = 3.4 Hz, C1-βH), 4.70 (d, 1H, Jgem = 12.6 Hz, Ph-CH ₂ a), 4.49 (d, 1H, Jgem = 12.6 Hz, Ph-CH ₂ b), 4.18-4.11 (m, 1H), 3.90-3.81 (m, 1H), 3.78-3.65 (m, 3H), 3.51 (t, 1H, J = 8.9 Hz), 1.85 (s, 3H, NHCOCH3 ₎ .
LCMS m/z: 400.1 [M+H] ⁺ . (Exact Mass: 399.17)
Purity (LCMS): 92.27%

(Synthesis of 2-(2-(R)-(2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionic acid (compound 4))

Benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α-D-glucopyranoside (compound 3) (13.07 g, 32.72 mmol) was suspended in toluene (220 mL) and concentrated to dryness under reduced pressure (60°C), and the residue was dissolved in dehydrated DMF (220 mL). NaH (60% in oil) (8.18 g, net 4.91 g, 204.5 mmol, 6.25 eq.) was added with stirring at room temperature under argon gas flow and stirred for 30 minutes. (S)-(-)-2-chloropropionic acid (10.00 g, 92.15 mmol, 2.8 eq.) was added dropwise to the reaction mixture over 25 minutes with stirring at room temperature under argon gas atmosphere and stirred for 30 minutes. Further, NaH (60% in oil) (8.18 g) was added under stirring at room temperature under argon gas atmosphere and stirred for 19 hours. Deionized water (33 mL) was carefully added to the reaction mixture while stirring at room temperature, and the mixture was concentrated to dryness under reduced pressure. The residue was suspended in water (500 mL) and adjusted to pH 3-4 with 2 mol/L hydrochloric acid (83 mL) while stirring at room temperature. The suspension was filtered, washed with water, dried by suction, dissolved in CHCl ₃ , dried over Na ₂ SO ₄ , filtered, and concentrated to dryness under reduced pressure to obtain a pale yellowish brown solid residue. The residue was crystallized from n-Hex./CHCl ₃ and dried (50°C, reduced pressure) to obtain compound 4 (10.11 g, 65.5%) as a white solid.
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ ppm: 8.11 (br, 1H, NH; D ₂ O exchange), 7.47-7.26 (m, 10H, Ph-H X2), 5.70 (s, 1H, PhCH), 5.05 (d, 1H, J = 3.2 Hz, C1-βH), 4.70 (d, 1H, Jgem = 12.4 Hz, Ph-CH ₂ a), 4.49 (d, 1H, Jgem = 12.4 Hz, Ph-CH ₂ b), 4.27 (q, 1H, J = 6.9 Hz, CH-CH ₃ ), 4.18-4.11 (m, 1H), 3.83-3.66 (m, 5H), 1.85 (s, 3H, NHCOCH ₃ ), 1.27 (d, 3H, J = 6.9 Hz, CH ₃ ).
LCMS m/z: 472.2 [M+H] ⁺ . (Exact Mass: 471.19)
Purity (LCMS): 97.25%

(Synthesis of (S)-methyl 2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionate (compound 5))

2-(2-(R)-(2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionic acid (compound 4) (1.89 g, 4.01 mmol) was dissolved in dehydrated DMF (40 mL), and N-methylmorpholine (1.32 mL, 12.03 mmol, 3.0eq.) and HBTU (3.04 g, 8.02 mmol, 2.0eq.) were added under an argon gas atmosphere and stirred at room temperature for 10 minutes. Then, L-alanine methyl ester hydrochloride (1.12 g, 8.02 mmol, 2.0eq.) was added and stirred for 17 hours. Water (36 mL) was added to the reaction mixture, which was then diluted with AcOEt and separated. The AcOEt layer was separated, washed with 1 mol/L HCl, saturated aqueous _NaHCO3 , and saturated aqueous NaCl, dried _{over Na2SO4} , filtered, and concentrated to dryness under reduced pressure to obtain a residue in the form of agar. The residue was purified by silica gel column chromatography (2% to 5% MeOH/ _CHCl3 :MeOH) and dried (60°C, reduced pressure) to obtain compound 5 (2.05 g, 91.9%) as a white solid.
^1H -NMR (400 MHz, DMSO-d ₆ ) δ ppm: 8.11 (d, 1H, J = 7.6 Hz, NHCO; D ₂ O exchange), 7.81 (d, 1H, J = 7.1 Hz, CONH; D ₂ O exchange), 7.45-7.27 (m, 10H, Ph-5H X 2), 5.71 (s, 1H, PhCH), 4.94 (d, 1H, J = 3.4 Hz, C1-βH), 4.71 (d, 1H, Jgem = 12.4 Hz, Ph-CH ₂ a), 4.51 (d, 1H, Jgem = 12.4 Hz, Ph- CH ₂ b), 4.33-4.12 (m, 3H), 4.03-3.93 (m, 1H), 3.85-3.64 (m, 4H), 3.58 (s, 3H, CO ₂ CH ₃ ), 1.82 (s, 3H, NHCO CH ₃ ), 1.29 (d, 3H, J = 7.1 Hz, CH ₃ ), 1.22 (d, 3H, J = 6.6 Hz, _CH3 ).
LCMS m/z: 557.7 [M+H] ⁺ . (Exact Mass: 556.24)
Purity (LCMS): 100.00%

　(Synthesis of (S)-2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionic acid (compound 7) (benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-OH))

(S)-Methyl 2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionate (compound 5) (1.90 g, 3.41 mmol) was suspended in MeOH (190 mL), 0.5 mol/L KOH (32 mL) was added, and the mixture was stirred at room temperature for 5 hours. After confirming the disappearance of the raw materials by TLC (10% MeOH/CHCl ₃ ), the reaction solution was concentrated to dryness under reduced pressure to obtain a white solid. The white solid was suspended in AcOEt (500 mL), and 1 mol/L HCl (100 mL) was added to separate the mixture. The AcOEt layer was separated, washed with saturated NaCl water, dried over Na ₂ SO ₄ , filtered, concentrated to dryness under reduced pressure, and then dried (60° C., reduced pressure) to obtain compound 7 (1.85 g, quant.) as a white solid.
^1H -NMR (400 MHz, DMSO-d ₆ ) δppm: 13.01-12.30 (br, 1H, COOH; D ₂ O exchange), 8.14 (d, 1H, J = 8.0 Hz, NHCO; D ₂ O exchange), 7.64 (d, 1H, J = 7.6 Hz, CONH; D ₂ O exchange), 7.48-7.25 (m, 10H, Ph- _H Jgem = 12.6 Hz, Ph- CH ₂ b), 4.27-4.10 (m, 3H), 4.04-3.93 (m, 1H), 3.83-3.64 (m, 4H), 1.81 (s, 3H, NHCO CH ₃ ), 1.27 (d, 3H, J= 7.3 Hz, CH ₃ ), 1.81 (d, 3H, J= 6.6 Hz, _CH3 ).
LCMS m/z: 543.7 [M+H] ⁺ . (Exact Mass: 542.23)
Purity (LCMS): 87.46%

(Synthesis of (R)-methyl 2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionate (compound 6))

D-Alanine methyl ester hydrochloride was condensed using 2-(2-(R)-(2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionic acid (Compound 4) (945 mg, 2.01 mmol) in the same manner as for Compound 5 to give Compound 6 (778 mg, 69.7%) as a white solid.
^1H -NMR (400 MHz, DMSO- _d6 ) δ ppm: 8.20 (d, 1H, J = 7.3 Hz, NHCO; _D2O exchange), 7.91 (d, 1H, J = 6.9 Hz, CONH; D2O exchange), 7.47-7.26 ₍ m, 10H, benzyl-Ph, benzylidene-Ph), 5.70 (s, 1H, benzylidene-CH), 4.99 (d, 1H, J = 3.7 Hz, MurNAc C1-βH), 4.71 (d, 1H, Jgem = 12.4 Hz, Ph- _CH2a ), 4.51 (d, 1H, Jgem = 12.4 Hz, Ph- _CH2b ), 4.30-4.12 (m, 3H), 3.97-3.88 (m, 1H), 3.83-3.58 (m, 4H), 3.61 (s, 3H, CO2 CH ₃ ), 1.87 (s, 3H, NHCO CH ₃ ), 1.28 (d, 3H, J = 7.1 Hz, MurNAc- CH ₃ or D-Ala- CH ₃ ), 1.24 (d, 3H, J = 6.6 Hz, MurNAc- CH ₃ or D-Ala- CH ₃ ),
LCMS m/z: 557.6 [M+H] ⁺ . (Exact Mass: 556.24)
Purity (LCMS): 94.89%

　(Synthesis of (R)-2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionic acid (compound 8) (benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-OH))

(R)-Methyl 2-((R)-2-((2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-3-α-D-glucopyranosyl)oxy)propionamido)propionate (compound 6) (750 mg, 1.35 mmol) was hydrolyzed with 0.5 mol/L KOH in MeOH solvent in the same manner as for compound 7 to obtain compound 8 (755 mg, quant.) as a white solid.
^1H -NMR (400 MHz, DMSO-d ₆ ) δ ppm: 12.70 (s, 1H, COOH; D ₂ O exchange), 8.23 (d, 1H, J = 7.1 Hz, NHCO; D ₂ O exchange), 7.78 (d, 1H, J = 7.1 Hz, CONH; D ₂ O exchange), 7.46-7.27 (m, 10H, benzyl-Ph, benzylidene-Ph), 5.70 (s, 1H, benzylidene-CH), 4.97 (d, 1H, J = 3.7 Hz, MurNAc C1-βH), 4.70 (d, 1H, Jgem = 12.4 Hz, Ph- CH ₂ a), 4.51(d, 1H, Jgem = 12.4 Hz, Ph- CH ₂ b), 4.31-4.11 (m, 3H), 3.96-3.87 (m, 1H), 3.83-3.61 (m, 4H), 1.86 (s, 3H, NHCO CH ₃ ), 1.28 (d, 3H, J = 7.3 Hz, MurNAc- CH ₃ or D-Ala- CH ₃ ), 1.24 (d, 3H, J = 6.9 Hz, MurNAc- CH ₃ or D-Ala- CH ₃ ).
LCMS m/z: 543.6 [M+H] ⁺ . (Exact Mass: 542.23)
Purity (LCMS): 96.51%

(Synthesis of 7-azido-4-methylcoumarin (compound 10))

7-Amino-4-methylcoumarin (4.00 g, 22.8 mmol) was suspended in 2 mol/L HCl aq. (40 mL), and NaNO ₂ (1.89 g, 27.4 mmol, 1.2 eq.) was added while stirring under ice cooling, and the mixture was stirred for 50 minutes. NaN ₃ (2.23 g, 34.2 mmol, 1.5 eq.) was then added, and the mixture was stirred for 1 hour. The precipitated solid was collected by filtration, washed with water, and dried (50°C, reduced pressure) to obtain compound 10 (4.54 g, 98.8%) as a pale ochre solid.
^1H -NMR (400 MHz, DMSO-d ₆ ) δppm: 7.79 (d, 1H, J = 8.5 Hz, C5-H), 7.17 (d, 1H, J = 2.3 Hz, C8-H), 7.15 (dd, 1H, J = 2.3, 8.5 Hz, C6-H), 6.35 (d, 1H, J = 1. 1 Hz, C3-H), 2.42 (d, 3H, J = 1.1 Hz, C4- CH ₃ ).
LCMS m/z: 202.4 [M+H] ⁺ . (Exact Mass: 201.05)
Purity (LCMS): 99.09%

(Synthesis of 1-azido-4-nitrobenzene (compound 22))

 

4-Nitroaniline hydrochloride (3.49 g, 20.0 mmol) was suspended in 6 mol/L HCl aq. (13 mL), and an aqueous solution (7 mL) of NaNO ₂ (1.40 g, 20.3 mmol, 1.02 eq.) was added with stirring under ice cooling, and the mixture was stirred for 5 minutes. Then, an aqueous solution (7 mL) of CH ₃ CO ₂ Na (1.12 g, 13.7 mmol, 0.69 eq.) and NaN ₃ (1.31 g, 20.1 mmol, 1.01 eq.) were added, and the mixture was stirred for 1 hour. The precipitated solid was filtered, washed with water, and dried (50°C, reduced pressure) to obtain compound 22 (2.58 g, 79.2%) as a yellow solid.
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 8.26 (d, 2H, J = 9.2 Hz), 7.36 (d, 2H, J = 9.2 Hz).
Purity (LCMS): 98.81%

(Synthesis of 4-methylcoumarin-7-amide (MCA) and p-nitroanilide (pNA) forms; synthesis of D-Glu(OBn)-MCA, D-Glu(OAll)-MCA, D-Glu(MCA)-OAll, D-Gln-MCA, D-isoGln-MCA and D-isoGln-pNA (general synthesis method))

(Preparation of Sodium Hydroselenide (NaHSe))
Degassed and dried IPA (130 mL/g Ce) was added to Se (1.2 eq.) and NaBH ₄ (1.44 eq.), and the mixture was stirred at room temperature under an argon gas atmosphere for 1 to 2 hours to prepare NaHSe (prepared just before use).

(Preparation of mixed acid anhydride)
N-Protected amino acid (1.2 eq.) was dissolved in dehydrated THF (130 mL/g), N-methylpiperidine (1.2 eq.) was added, and isopropyl chloroformate (ca. 2 mol/L in toluene) (1.4 eq.) was added with stirring at -15°C under an argon gas atmosphere, and the mixture was stirred for 30 minutes to 1 hour.

(Synthesis of N-protected amino acid 4-methylcoumarin-7-amide (MCA form) and p-nitroanilide (pNA form))
The above mixed acid anhydride was slowly added to a freshly prepared solution of NaHSe in IPA at -15°C, and the mixture was stirred under argon gas for 30 minutes to 1 hour. Next, a solution of 7-azido-4-methyl-coumarin (1.0 eq.) in dehydrated THF (30 mL/g) or a solution of 1-azido-4-nitrobenzene (1.0 eq.) in dehydrated THF (18 mL/g) was added under argon gas while stirring at room temperature, and the mixture was stirred overnight at room temperature. The black suspension was removed by filtration using a Celite pad and washed with 20% MeOH/CHCl _3. The filtrate and washings were combined and concentrated to dryness under reduced pressure, the residue was dissolved in 20% MeOH/CHCl ₃ , silica gel (FL100 D) was added, and the mixture was concentrated to dryness under reduced pressure, and purified by silica gel column chromatography.

(Synthesis of Fmoc-D-Glu(OAll)-MCA (compound 13))

Degassed and dried IPA (120 mL) was added to Se (942 mg, 11.93 mmol, 1.2 eq.) and _NaBH4 (542 mg, 14.32 mmol, 1.44 eq.), and the mixture was stirred at room temperature under an argon gas atmosphere for 2.5 hours to prepare NaHSe (11.99 mmol, 1.4 eq.) (prepared just before use). Fmoc-D-Glu(OAll)-OH (compound 12) (4.88 g, 11.93 mmol, 1.2 eq.) was dissolved in dehydrated THF (120 mL), N-methylpiperidine (1.45 mL, 11.93 mmol, 1.2 eq.) was added, and isopropyl chloroformate (ca. 2 mol/L in toluene) (6.0 mL, 11.93 mmol, 1.4 eq.) was added with stirring at -15°C under argon gas atmosphere, and the mixture was stirred for 1.5 hours. The above mixed acid anhydride was slowly added to a freshly prepared IPA solution of NaHSe at -15°C, and the mixture was stirred for 25 minutes under argon gas atmosphere. Next, a solution of 7-azido-4-methylcoumarin (compound 10) (1.73 g, 8.60 mmol) in dehydrated THF (50 mL) was added under argon gas atmosphere with stirring at room temperature, and the mixture was stirred overnight at room temperature. The suspension was filtered off using a Celite pad and washed with 20% MeOH/CHCl _3. The filtrate and washings were combined and concentrated to dryness under reduced pressure. The residue was dissolved in 20% MeOH/CHCl ₃ , silica gel (FL100D; 20 g) was added, and the mixture was concentrated to dryness under reduced pressure. The product was purified by silica gel column chromatography (CHCl ₃ to 15% AcOEt/CHCl ₃ ). The fractions with Rf = 0.39 (25% AcOEt/CHCl ₃ ) were combined and concentrated to dryness under reduced pressure. The residue was washed with n-Hex./CHCl ₃ to give compound 13 (2.20 g, 45.1%) as a pale yellow solid.
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.53 (s, 1H, Coumarin C7-NH; D ₂ O exchange), 7.90 (d, 2H, J = 7.6 Hz, Fmoc-arom.), 7.84 (d, 1H, Glu-NH; D ₂ O exchange), 7.80-7.70 (m, 4H, Fmoc-arom2H, Coumarin C5-H & C8-H [7.78 (d, 1H, J = 2.1 Hz]), 7.51 (dd, 1H, J = 2.1, 8.7 Hz, Coumarin C6-H), 7.46-7.29 (m, 2H, Fmoc-arom.), 6.28(d, 1H, J = 1.1 Hz, Coumarin C3-H), 5.96-5.83 (m, 1H, O- CH ₂ -CH= CH ₂ ), 5.33-5.25 (m, 1H, O- CH ₂ -CH= CH ₂ a), 5.23-5.16 (m, 1H, O- CH ₂ -CH= CH ₂ b), _{Coumarin C4- ​ ​ CH3} ), 2.12-1.87 (m, 2H, Glu-γCH ₂ a,b).
LCMS m/z: 567.5 [M+H] ⁺ . (Exact Mass: 566.21)
Purity (LCMS): 99.58%

(Synthesis of Fmoc-D-Glu(MCA)-OAll (compound 15))

Fmoc-D-Glu(OH)-OAll (compound 14) (4.72 g) was reacted and worked up according to the general synthetic method to obtain Fmoc-D-Glu(MCA)-OAll (compound 15) (2.47 g, 43.8%) as a yellow solid.
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 10.38 (s, 1H, coumarin C7-NH; _D2O exchange), 7.94-7.84 (m, 3H, Fmoc-arom2H, Glu-NH; _D2O exchange), 7.77-7.67 (m, 4H, Fmoc-arom2H, coumarin C5-H & C8-H [7.75 (d, 1H, J = 2.1 Hz; coumarin C8-H), 7.69 (d, 1H, J = 8.7 Hz, coumarin C5-H)]), 7.47 (dd, 1H, J = 2.1, 8.7 Hz, coumarin C6-H), 7.45-7.39 (m, 2H, Fmoc-arom.), 7.37-7.30 (m, 2H, Fmoc-arom.), 6.24 (d, 1H, J = 1.1 Hz, Coumarin C3-H), 5.97-5.86 (m, 1H, O- CH ₂ -CH= CH ₂ ), 5.37-5.28 (m, 1H, O- CH ₂ -CH= CH ₂ a), 5.24-5.18 (m, 1H, O- CH ₂ -CH= CH ₂ b), 4.65-4.56 (m, 2H, O- CH ₂ -CH= CH ₂ ), 4.38-4.11 (m, 4H, Fmoc-CH, CH ₂ & Glu-αCH), 2.56-2.47 (m, 2H, Glu-βCH ₂ ), 2.38 (d, 3H, J = 1.1 Hz, Coumarin C4- CH ₃ ), 2.21-2.07 (m, 1H, Glu-γCH ₂ a), 1.99-1.85 (m, 1H, Glu-γCH ₂ b).
LCMS m/z: 567.5 [M+H] ⁺ . (Exact Mass: 566.21)
Purity (LCMS): 99.44%

(Synthesis of Fmoc-D-Gln-MCA (compound 19))

Fmoc-D-Gln-OH (compound 18) (4.41 g) was reacted and worked up according to the general synthetic method to obtain Fmoc-D-Gln-MCA (compound 19) (477 mg, 9.1%) as a pale yellow solid.
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 10.53 (s, 1H, coumarin C7-NH; _D2O exchange), 7.90 (d, 2H, J = 7.6 Hz, Fmoc-arom.), 7.82 (d, 1H, J = 7.6 Hz, OCONH; _D2O exchange), 7.80 (d, 1H, J = 2.1 Hz, coumarin C8-H), 7.78-7.72 (m, 3H, Fmoc-arom2H, coumarin C5-H), 7.51 (dd, 1H, J = 2.1, 8.7 Hz, coumarin C6-H), 7.46-7.39 (m, 2H, Fmoc-arom.), 7.38-7.30 (m, 3H, Fmoc-arom.2H, CONH ₂ a; D ₂ O exchange), 6.84 (s, 1H, CONH ₂ b; D ₂ O exchange), 6.28 (d, 1H, J = 1.1 Hz, Coumarin C3-H), 4.31-4.20 (m, 3H, Fmoc-CH, CH ₂ ), 4.19-4.11 (m, 1H, Gln-αH), 2.41 (d, 3H, J = 1.1 Hz, Coumarin C4- CH ₃ ), 2.29-2.10 (m, 2H, Gln-βCH ₂ ), 2.03-1.92 (m, 1H, Gln-γCH ₂ a), 1.91-1.80 (m, 1H, Gln-γCH ₂ b).
LCMS m/z: 526.6 [M+H] ⁺ . (Exact Mass: 525.19)
Purity (LCMS): 97.60%

(Synthesis of Fmoc-D-isoGln-MCA (compound 21))

Fmoc-D-isoGln-OH (compound 20) (3.67 g) was reacted and worked up according to the general synthetic method to obtain Fmoc-D-isoGln-MCA (compound 21) (1.24 g, 28.4%) as a white to pale yellow solid.
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 10.38 (s, 1H, coumarin C7-NH; _D2O exchange), 7.89 (d, 2H, J = 7.6 Hz, Fmoc-arom.), 7.76 (d, 1H, J = 1.8 Hz, coumarin C8-H), 7.73 (d, 2H, J = 7.3 Hz, Fmoc-arom.), 7.69 (d, 1H, J = 8.7 Hz, coumarin C5-H), 7.51-7.38 (m, 4H, CONHCH; _D2O exchange, Fmoc-arom.2H, coumarin C6-H), 7.36 (s, 1H, _CONH2a ; _D2O exchange), 7.32 (t, 2H, J = 7.3 Hz, Fmoc-arom.), 7.09 (s, 1H, CONH ₂ b; D ₂ O exchange), 6.25 (d, 1H, J = 1.1 Hz, Coumarin C3-H), 4.30-4.16 (m, 3H), 4.04-3.95 (m, 1H), 2.44 (t, 2H, J = 7.8 Hz, isoGln-βCH ₂ ), 2.38 (d, 3H, J = 1.1 Hz, Coumarin C4- CH ₃ ), 2.11-1.97 (m, 1H, isoGln-γCH ₂ a), 1.93-1.79 (m, 1H, isoGln-γCH ₂ b).
LCMS m/z: 526.6 [M+H] ⁺ . (Exact Mass: 525.19)
Purity (LCMS): 97.84%

(Synthesis of Fmoc-D-isoGln-pNA (compound 23))

Fmoc-D-isoGln-OH (compound 20) (4.40 g) was reacted and worked up according to the general synthetic method to obtain Fmoc-D-isoGln-pNA (compound 23) (2.98 g, 61.5%) as a pale yellow solid.
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.56 (s, 1H, pNA-NH; D ₂ O exchange), 8.20 (d, 2H, J = 9.2 Hz, pNA C3,5-H), 7.89 (d, 2H, J = 7.6 Hz, Fmoc-arom.), 7.83 (d, 2H, J = 9.2 Hz, pNA C2,6-H), 7.73 (d, 2H, J = 7.6 Hz, Fmoc-arom.), 7.46 (d, 1H, J = 8.2 Hz, isoGln-NH; D ₂ O exchange), 7.42 (t, 2H, J = 7.6 Hz, Fmoc-arom.), 7.36(s, 1H, CONH ₂ a; D ₂ O exchange), 7.33 (t, 2H, J = 7.6 Hz; Fmoc-arom.), 7.09 (s, 1H, CONH ₂ b; D ₂ O exchange), 4.33-4.16 (m, 3H, Fmoc-CH, CH ₂ ), 3.99 (dt, 1H, J = 5.0, 8.7 Hz, isoGln-αH), 2.47 (t, 2H, J = 7.8 Hz, isoGln-βCH ₂ ), 2.11-1.96 (m, 1H, isoGln-γCH ₂ a), 1.93-1.79 (m, 1H, isoGln-γCH ₂ b).
LCMS m/z: 489.6 [M+H] ⁺ . (Exact Mass: 488.17)
Purity (LCMS): 98.67%

<Fmoc-L-Ala conversion (general method)>
Fmoc-L-Ala-OH (hydrate) (compound 33) (1.1 eq.) was dissolved in dehydrated DMF (40 mL/g), and _HOBt.H2O (1.3 eq.) and EDCI (1.3 eq.) were added with stirring at 0°C and stirred for 30 minutes. Next, DIEA (2.2 eq.) and HD-Glu(MCA)-OAll etc. (compound 27 etc.) (1.0 eq.) were added to the reaction mixture and stirred for 1 hour, and further stirred at room temperature for 3 hours. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified to obtain the target compound.

(Synthesis of Fmoc-L-Ala-D-Glu(MCA)-OAll (compound 37))

HD-Glu(MCA)-OAll (compound 27) (952 mg, 2.76 mmol) was used in the reaction according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was added with AcOEt (200 mL) (both layers contained insoluble matter), washed with water, saturated NaHCO ₃ water, and then saturated NaCl water, and the AcOEt layer (containing suspended matter) was separated. It was dried over Na ₂ SO ₄ , filtered off, washed 2 and 3 times with 20% MeOH/CHCl _3, and the filtrate and washings were combined and concentrated under reduced pressure. The precipitate was collected by filtration, washed with n-Hex./AcOEt, and dried (50°C, reduced pressure) to obtain compound 37 (1.79 g, quant.) (Rf = 0.57, 0.64 [10%MeOH/ CHCl ₃ ]. The amount ratio is unknown because there are no separated peaks in the ¹ H-NMR spectrum).
Rf = 0.64 (principal component)
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 10.50 (s, 1H, coumarin-C7NH; _D2O exchange), 8.41 (d,1H, J = 7.8 Hz, NH; D2O exchange), 7.91-7.84 (m, 2H, Fmoc-arom. ₎ , 7.78-7.66 (m, 4H, Fmoc-arom2H, coumarin C5-H & C8-H [7.76 (d, 1H, J = 1.8 Hz, coumarin C8-H), 7.69 (d, 1H, J = 8.7 Hz, coumarin C5-H)]), 7.53 (d, 1H, J = 8.0 Hz, NH; _D2O exchange), 7.48 (dd, 1H, J = 1.8, 8.7 Hz, Coumarin C6-H), 7.44-7.37 (m, 2H, Fmoc-arom.), 7.33-7.28 (m, 2H, Fmoc-arom.), 6.25 (d, 1H, J = 1.1Hz, Coumarin C3-H), 5.97-5.84 (m, 1H, O- CH ₂ -CH= CH ₂ ), 5.36-5.27 (m, 1H, O- CH ₂ -CH= CH ₂ a), 5.23-5.16 (m, 1H, O- CH ₂ -CH= CH ₂ b), 4.62-4.54 (m, 2H, O- CH ₂ -CH= CH ₂ ), 4.38-4.29 (m, 1H), 4.28-4.09 (m, 4H), 2.52-2.45 (m, 2H, Glu-βCH ₂ ), 2.39 (d, 3H, J =1.1 Hz, Coumarin C4- CH ₃ ), 2.18-2.06 (m, 1H, Glu-γCH ₂ a), 2.01-1.88 (m, 1H, Gln-γCH ₂ b), 1.26 (d, 3H, J = 7.1 Hz, Ala- CH ₃ ).
LCMS m/z: 638.6 [M+H] ⁺ . (Exact Mass: 637.24)
Purity (LCMS): 99.21%

(Synthesis of Fmoc-L-Ala-D-Gln-MCA (compound 38))

HD-Gln-MCA (compound 26) (220 mg, 0.73 mmol) was used in the reaction according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was suspended in CHCl ₃ (10 mL), n-Hex. (10 mL) was added, and the suspension was filtered, washed with water, and dried (50°C, reduced pressure) to obtain compound 38 (374 mg; Rf = 0.15, 0.25 [10%MeOH/CHCl ₃ ] mixture) as a white solid. From ^{the 1} H-NMR spectrum, it was estimated to be a mixture of two isomers (abundance ratio about 4:1).
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 10.29 (s, 0.2H, coumarin C7-NH; _D2O exchange), 10.23 (s, 0.8H, coumarin C7-NH; _D2O exchange), 8.42 (d,1H, J = 8.0 Hz, NH; _D2O exchange), 7.86 (t, 2H, J = 6.9 Hz, Fmoc-arom.), 7.83 (d, 0.2H, J = 1.8 Hz, coumarin C8-H), 7.80 (d, 0.8H, J = 1.8 Hz, coumarin C8-H), 7.76-7.64 (m, 4H; 1H- _D2O exchange), 7.56 (dd, 0.2H, J = 2.1, 8.7 Hz, coumarin C6-H), 7.52 (dd, 0.8H, J = 2.1, 8.7 Hz, coumarin C6-H), 7.44-7.27 (m, 5H; 1H- D ₂ O exchange), 6.81 (br-s, 1H, CONH ₂ b), 6.28 (s, 1H, coumarin C3-H), 4.45-4.35 (m, 1H), 4.33-4.05 (m, 4H), 2.39 (s, 3H, coumarin C4- CH ₃ ), 2.18-1.96 (m, 3H, Gln-βCH ₂ , Gln-γCH ₂ a), 1.90-1.77 (m, 1H, Gln-γCH ₂ b), 1.26 (d, 2.4H, J = 7.1 Hz; Ala- CH ₃ ), 1.22 (d, 0.6H, J = 7.1 Hz; Ala- CH ₃ ).
LCMS m/z: 597.6 [M+H] ⁺ . (Exact Mass: 596.23)
Purity (LCMS): 96.36%

(Synthesis of Fmoc-L-Ala-D-isoGln-pNA (compound 40))

HD-isoGln-pNA (compound 31) (500 mg, 1.88 mmol) was used in the reaction according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was dissolved in 20% MeOH/CHCl ₃ , silica gel (FL100D 20 g) was added, concentrated to dryness, and purified by silica gel column chromatography (5% to 10%, MeOH/CHCl ₃ ). The target fractions were combined, concentrated to dryness, and dried (50°C, reduced pressure) to obtain compound 40 (914 mg, 87.0%) as a white solid. ^1H -NMR spectrum suggested that it was a mixture of two isomers (abundance ratio approximately 9:1).
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.53 (s, 1H, pNA-NH; D ₂ O exchange), 8.20 (d, 0.2H, J = 9.2 Hz, pNA C3,5-H), 8.19 (d, 1.8H, J = 9.2 Hz, pNA C3,5-H), 8.14 (d, 0.1H, J = 7.3 Hz, NH; D ₂ O exchange), 8.09 (d, 0.9H, J = 8.2 Hz, NH; D ₂ O exchange), 7.98 (d, 0.1H, J = 8.0 Hz, NH; D ₂ O exchange), 7.85 (d, 2H, J =7.3 Hz, Fmoc-arom.), 7.82 (d, 0.2H, J = 9.2Hz, pNA C2,6-H), 7.81 (d, 1.8H, J = 9.2 Hz, pNA C2,6-H), 7.72 (t, 2H, J = 6.9 Hz, Fmoc-arom.), 7.61 (d, 1H, J = 7.1 Hz, NH; D ₂ O exchange), 7.41 (t, 2H, J =6.9 Hz, Fmoc-arom.), 7.36-7.27 (m, 3H, Fmoc-arom. & isoGln CONH ₂ a; D ₂ O exchange), 7.16 (s, 1H, isoGln CONH ₂ b; D ₂ O exchange), 4.32-4.16 (m, 4H), 4.08 (dq, 1H, J = 7.1, 7.1 Hz, Ala-αH), 2.41 (t, 2H, J = 8.0 Hz, isoGln-βCH ₂ ), 2.16-2.00 (m, 1H, isoGln-γCH ₂ a), 1.91-1.75 (m, 1H, isoGln-γCH ₂ b), 1.24 (d, 0.3H, J = 7.1 Hz, Ala- CH ₃ ), 1.23 (d, 2.7H, J = 7.1 Hz, Ala- CH ₃ ).
LCMS m/z: 560.6 [M+H] ⁺ . (Exact Mass: 559.21)
Purity (LCMS): 100.00% (11.38%, 88.62%).

<De-Fmoc (general law)>
The Fmoc compound (1.0 eq.) was dissolved in 20% piperidine/DMF solution (22 mL/g) and stirred at room temperature for 20 minutes. After confirming the disappearance of the raw material by TLC (10% MeOH/CHCl ₃ ), the reaction solution was concentrated to dryness under reduced pressure, and the residue was purified to obtain the target compound.

(Synthesis of H-D-Glu(OAll)-MCA (compound 28) and H-D-Glu(OH)-MCA (compound 29))

Fmoc-D-Glu(OAll)-MCA (compound 13) (1.80 g, 3.18 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was reprecipitated with n-Hex. (30 mL)/CHCl ₃ (15 mL), stirred, filtered, and dried (60°C, reduced pressure) to obtain HD-Glu(OH)-MCA (compound 29) (680 mg) as a pale yellow solid. The filtrate was concentrated to dryness under reduced pressure, dissolved in CHCl ₃ , and purified by silica gel column chromatography (CHCl ₃ to 8%MeOH/CHCl ₃ ). The fraction with Rf = 0.40 (10%MeOH/CHCl ₃ ) was concentrated to dryness under reduced pressure, and the residue was washed with n-Hex./CHCl ₃ to obtain compound 28 (110 mg, 11.0%) as a pale yellow solid. In addition, the fraction with Rf = 0.28 (10% MeOH/CHCl ₃ ) was concentrated to dryness under reduced pressure, and the residue was washed with n-Hex./CHCl ₃ to obtain compound 29 (132 mg; total yield: 812 mg, 74.2%) as a slightly yellow to white solid.
HD-Glu(OAll)-MCA:
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 7.83 (d, 1H, J = 2.1 Hz, coumarin C8-H), 7.72 (d, 1H, J = 8.7 Hz, coumarin C5-H), 7.57 (dd, 1H, J = 2.1, 8.7 Hz, coumarin C6-H), 6.28 (d, 1H, J = 1.1 Hz, coumarin C3-H), 5.98-5.84 (m, 1H, O- _CH2 -CH= _CH2 ), 5.34-5.26 (m, 1H, O- _CH2 -CH= _CH2a ), 5.23-5.17 (m, 1H, O- _CH2 -CH= _CH2b ), 4.55-4.52 (m, 2H, O- CH ₂ -CH= CH ₂ ), 3.36 (dd, 1H, J = 5.3, 8.2 Hz, Glu-αH), 2.53-2.43 (m, 2H, Glu-βCH ₂ ), 2.40 (d, 3H, J = 1.1 Hz, Coumarin C4- CH ₃ ), 2.02-1.90 (m, 1H, Glu-γCH ₂ a), 1.80-1.66 (m, 1H, Glu-γCH ₂ b).
LCMS m/z: 345.5 [M+H] ⁺ . (Exact Mass: 344.14)
Purity (LCMS): 98.961% (71.48% + 27.48% [M-OAll])
HD-Glu(OH)-MCA:
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 10.50 (s, 1H, coumarin C7-NH; _D2O exchange), 7.93 (s, 1H, COOH; _D2O exchange), 7.78 (d, 1H, J = 2.1 Hz, coumarin C8-H), 7.74 (d, 1H, J = 8.7 Hz, coumarin C5-H), 7.53 (dd, 1H, J = 2.1, 8.7 Hz, coumarin C6-H), 6.28 (d, 1H, J = 1.1 Hz, coumarin C3-H), 4.23 (dd, 1H, J = 4.4, 8.7 Hz, Glu-αH), 2.44-2.32 (m, 1H), 2.40 (d, 3H, J = 1.1 Hz, coumarin C4- _CH3 ), 2.31-2.11 (m, 2H), 2.08-1.97 (m, 1H).
LCMS m/z: 287.5 [M-OH]+. (Exact Mass: 304.11)
Purity (LCMS): 100.00%

(Synthesis of H-D-Glu(MCA)-OAll (compound 27))

Fmoc-D-Glu(MCA)-OAll (compound 15) (1.80 g, 3.18 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was reprecipitated with n-Hex. (30 mL)/CHCl ₃ (30 mL), stirred, filtered, and dried (50°C, reduced pressure) to obtain compound 27 (983 mg, 89.9%) as a pale yellow solid.
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 10.38 (s, 1H, coumarin C7-NH; _D2O exchange), 7.76 (d, 1H, J = 2.1 Hz, coumarin C8-H), 7.70 (d, 1H, J = 8.7 Hz, coumarin C5-H), 7.48 (dd, 1H, J = 2.1, 8.7 Hz, coumarin C6-H), 6.25 (d, 1H, J = 1.1 Hz, coumarin C3-H), 6.00-5.87 (m, 1H, O- _CH2 -CH= _CH2 ), 5.37-5.29 (m, 1H, O- _CH2 -CH= _CH2a ), 5.25-5.19 (m, 1H, O- CH ₂ -CH= CH ₂ b), 4.61-4.56 (m, 2H, O- CH ₂ -CH= CH ₂ ), 3.39 (dd, 1H, J = 5.3, 8.5 Hz, Glu-αH), 2.52-2.47 (m, 2H, Glu-βCH ₂ ), 2.39 (d, 3H, J = 1.1 Hz, coumarin C4- CH ₃ ), 2.04-1.92 (m, 1H, Glu-γCH ₂ a), 1.92-1.79 (br, 2H, NH ₂ ; D ₂ O exchange), 1.79-1.67 (m, 1H, Glu-γCH ₂ b).
LCMS m/z: 345.5 [M+H] ⁺ . (Exact Mass: 344.14)
Purity (LCMS): 97.36%

(Synthesis of H-D-Gln-MCA (compound 26))

Fmoc-D-Gln-MCA (compound 19) (450 mg, 0.86 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was suspended in n-Hex. (10 mL)/CHCl ₃ (10 mL), stirred, and filtered. The filtered product was dried (50°C, reduced pressure) to obtain compound 26 (249 mg, 95.8%) as a pale ochre solid.
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 7.85 (d, 1H, J = 2.1 Hz, coumarin C8-H), 7.73 (d, 1H, J = 8.7 Hz, coumarin C5-H) _, 7.57 (dd, 1H, J = 2.1, 8.7 Hz, _coumarin C6-H), 7.30 (br-s, _1H , CONH2a; _D2O exchange), 6.76 (br-s, 1H, CONH2b; D2O exchange), 6.27 (d, 1H, J = 1.1 Hz, coumarin C3-H), 3.41-3.29 (m, 1H, Gln-αH), 2.40 (d, 3H, J = 1.1 Hz, Coumarin C4- CH ₃ ), 2.25-2.09 (m, 2H, Gln-βCH ₂ ), 1.94-1.82 (m, 1H, Gln-γCH ₂ a), 1.73-1.61 (m, 1H, Gln-γCH ₂ b).
LCMS m/z: 304.5 [M+H] ⁺ . (Exact Mass: 303.12)
Purity (LCMS): 74.88%

(Synthesis of H-L-Ala-D-Gln-MCA (compound 44))

Fmoc-L-Ala-D-Gln-MCA (compound 38) (350 mg, 0.59 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was suspended in n-Hex. (20 mL)/CHCl ₃ (10 mL), stirred, and filtered (precipitate; moisture absorption). The filtered product was dissolved in 20% MeOH/CHCl ₃ and concentrated to dryness under reduced pressure. The residue was powdered in n-Hex. and dried (50°C, reduced pressure) to obtain compound 44 (213 mg, 96.8%) as a white solid. ^1H -NMR spectrum suggested that it was a mixture of two isomers (abundance ratio about 5:1).
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 10.53 (s, 0.84H, coumarin C7-NH; _D2O exchange), 10.31 (s, 0.16H, coumarin C7-NH; D2O exchange), 8.41-7.91 (br, 1H, NH; _D2O exchange), 7.83 (d, 0.16H, J = 2.1 Hz, coumarin C8-H), 7.78 (d, 0.84H, _J = 2.1 Hz, coumarin C8-H), 7.74 (d, 1H, J = 8.7 Hz, coumarin C5-H), 7.59 (dd, 0.16H, J = 2.1, 8.7 Hz, Coumarin C6-H), 7.50 (dd, 0.84H, J = 2.1, 8.7 Hz, Coumarin C6-H), 7.32 (br-s, 1H, CONH ₂ a; D ₂ O exchange), 6.79 (br-s, 1H, CONH ₂ b; D ₂ O exchange), 6.28 (d, 1H, J = 1.1 Hz, Coumarin C3-H), 4.42 (br-dd, 1H, J = 5.5, 8.0 Hz, Gln-αH), 3.35 (q, 0.84H, J = 6.9 Hz, Ala-αCH), 3.30 (q, 0.16H, J = 6.9 Hz, Ala-αCH), 2.40 (d, 3H, J = 1.1 Hz, Coumarin C4- CH ₃ ), 2.20-2.07 (m, 2H, Gln-βCH ₂ ), 2.03-1.81 (m, 2H, Gln-γCH ₂ ), 1.14 (d, 2.52H, J = 6.9 Hz, Ala- CH ₃ ), 1.13 (d, 0.48H, J = 6.9 Hz, Ala- CH ₃ ).
LCMS m/z: 375.6 [M+H] ⁺ . (Exact Mass: 374.16)
Purity (LCMS): 94.76% (83.03%, 11.73%).

(Synthesis of H-D-isoGln-MCA (compound 30))

Fmoc-D-isoGln-MCA (compound 21) (1.14 g, 2.17 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was suspended in n-Hex. (25 mL)/CHCl ₃ (25 mL), stirred, and filtered. The filtered product was dried (60°C, reduced pressure) to obtain compound 30 (602 mg, 91.5%) as a white solid.
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 10.40 (br-s, 1H, CONH; _D2O exchange), 7.77 (d, 1H, J = 1.8 Hz, coumarin C8-H), 7.70 (d, 1H, J = 8.7 Hz, coumarin C5-H), 7.48 (dd, 1H, J = 2.1, 8.7 Hz, coumarin C6-H), 7.33 (br-s, 1H, _CONH2a ; _D2O exchange), 6.97 (br-s, 1H, _CONH2b ; _D2O exchange), 6.25 (d, 1H, J = 1.1 Hz, coumarin C3-H), 3.17-3.11 (m, 1H, isoGln-αH), 2.44 (t, 2H, J = 7.8 Hz, isoGln-γCH ₂ ), 2.39 (d, 3H, J = 1.1 Hz, coumarin C4- CH ₃ ), 1.94-1.83 (m, 1H, isoGln-βCH ₂ a), 1.80 (br, 2H, isoGln-αNH ₂ ; D ₂ O exchange), 1.73-1.65 (m, 1H, isoGln-βCH ₂ b).
LCMS m/z: 304.5 [M+H] ⁺ . (Exact Mass: 303.12)
Purity (LCMS): 97.81%

(Synthesis of H-L-Ala-D-isoGln-MCA・HCl (compound 45))

Fmoc-L-Ala-D-isoGln-MCA (compound 39) (80 mg, 0.13 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was suspended in n-Hex. (15 mL)/CHCl ₃ (7 mL), stirred, and filtered. The filtered product was dried (50°C, reduced pressure) to obtain HL-Ala-D-isoGln-MCA (52 mg, quant.) as a pale ochre solid.
LCMS m/z: 375.2 [M+H] ⁺ . (Exact Mass: 374.16), Purity (LCMS): 95.65%
HL-Ala-D-isoGln-MCA (50 mg, 0.13 mmol) was dissolved in a mixture of CHCl ₃ (10 mL)/MeOH (5 mL), and 5% HCl/MeOH (0.2 mL) was added and mixed. The mixture was concentrated to dryness under reduced pressure to obtain a pale ochre solid residue. The residue was washed with n-Hex. (5 mL)/CHCl ₃ (5 mL) and dried (50°C, reduced pressure) to obtain compound 45 (52 mg, 94.5%).
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 10.52 (s, 1H, coumarin C7-NH; _D2O exchange), 8.62 (d, 1H, J = 8.0 Hz, isoGln-NH; _D2O exchange), 7.96-7.41 (br, 3H, Ala- _NH3 ⁺ ; _D2O exchange), 7.78 (d, 1H, J = 1.8 Hz, coumarin C8-H), 7.71 (d, 1H, J = 8.7 Hz, coumarin C5-H), 7.53 (s, 1H, _CONH2a ; _D2O exchange), 7.50 (dd, 1H, J = 1.8, 8.7 Hz, coumarin C6-H), 7.19 (s, 1H, CONH ₂ b; D ₂ O exchange), 6.26 (d, 1H, J = 1.1 Hz, coumarin C3-H), 4.34-4.23 (m, 1H, Ala-αCH), 3.91-3.80 (m, 1H, isoGln-αH), 2.43 (t, 2H, J = 8.0 Hz, isoGln-βCH ₂ ), 2.40 (d, 3H, J = 1.1 Hz, Coumarin C4- CH ₃ ), 2.15-2.03 (m, 1H, isoGln-γCH ₂ a), 1.94-1.80 (m, 1H, isoGln-γCH ₂ b), 1.35(d, 3H, J = 6.9 Hz, Ala- _CH3 ).
LCMS m/z: 375.3 [M+H] ⁺ . (Exact Mass: 374.16)
Purity (LCMS): 95.46%

(Synthesis of H-D-isoGln-pNA (compound 31))

Fmoc-D-isoGln-pNA (compound 23) (2.96 g, 6.06 mmol) was reacted according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was crystallized from n-Hex. (60 mL)/CHCl ₃ (30 mL), filtered, and dried (50°C, reduced pressure) to obtain compound 31 (1.51 g, 93.5%) as a pale gray solid.
^1H -NMR (400 MHz, DMSO-d ₆ ) δppm: 11.18-10.01 (br, 1H, pNA-NH; D ₂ O exchange), 8.21 (d, 2H, J = 9.4 Hz, pNA C3,5-H), 7.84 (d, 2H, J = 9.4 Hz, pNA C2,6-H), 7.34 (s, 1H, CONH ₂ a; D ₂ O exchange), 6.98 (s, 1H, CONH ₂ b; D ₂ O exchange), 3.14 (dd, 1H, J = 5.5, 7.8 Hz, isoGln-αH), 2.47 (t, 2H, J = 7.8 Hz, isoGln-βCH ₂ ), 2.04-1.53 (m, 4H [2.04-1.53 (br, 2H, NH ₂ ; D ₂ O exchange), 1.94-1.82 (m, 1H, isoGln-γCH ₂ a), 1.76-1.63 (m, 1H, isoGln-γCH ₂ b]).
LCMS m/z: 267.5 [M+H] ⁺ . (Exact Mass: 266.10)
Purity (LCMS): 97.63%

(Synthesis of H-L-Ala-D-isoGln-pNA (compound 46))

Fmoc-L-Ala-D-isoGln-pNA (compound 40) (885 mg, 1.58 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. n-Hex. (15 mL)/CHCl ₃ (15 mL) was added to the residue, which was stirred, filtered, and dried (50°C, reduced pressure) to obtain compound 46 (474 mg, 88.8%) as a white solid. From the ¹ H-NMR spectrum, it was estimated to be a mixture of two isomers (abundance ratio approximately 9:1).
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.56 (s, 0.9H, pNA-NH; D ₂ O exchange), 10.54 (s, 0.1H, pNA-NH; D ₂ O exchange), 8.21 (d, 2H, J = 9.4 Hz, pNA C3,5-H), 8.11-7.91 (br, 1H, D-isoGln-NH; D ₂ O exchange), 7.83 (d, 2H, J = 9.4 Hz, pNA C2,6-H), 7.47 (s, 0.9H, isoGln- CONH ₂ a; D ₂ O exchange), 7.38 (s, 0.1H, isoGln- CONH ₂ a; D ₂ O exchange), 7.15 (s, 0.9H, isoGln- CONH ₂ b; D ₂ O exchange), 7.12 (s, 0.1H, isoGln- CONH ₂ b; D ₂ O exchange), 4.26 (br-dd, 1H, J = 5.7, 7.3 Hz, isoGln-αH), 3.29 (q, 1H, J = 6.9 Hz, Ala-αH), 2.40 (t, 2H, J = 8.0 Hz, isoGln-βCH ₂ ), 2.14-1.73 (m, 4H, isoGln-γCH ₂ a,b, Ala- NH ₂ ; D ₂ O exchange), 1.122 (d, 2.7H, J = 6.9 Hz, Ala- CH ₃ ), 1.116(d, 0.3H, J = 6.9 Hz, Ala- CH ₃ ).
LCMS m/z: 338.5 [M+H] ⁺ . (Exact Mass: 337.14)
Purity (LCMS): 95.02% (84.76% + 10.26%)

<Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(OAll)-MCA and -D-Glu(MCA)-OAll (EDCI-HOBt method) (general method)>
Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-OH (compound 7) (1.1 eq.) was dissolved in dehydrated DMF (10 mL/g), HOBt.H ₂ O (1.3 eq.) and EDCI (1.3 eq.) were added, and the mixture was stirred for 30 minutes under ice cooling. DIEA (74 mg, 0.57 mmol, 2.2 eq.), HD-Glu(OAll)-MCA or HD-Glu(MCA)-OAll (1.0 eq.) were then added to the reaction mixture, and the mixture was stirred for 1 hour, and further stirred at room temperature for 2 hours. The reaction mixture was concentrated to dryness under reduced pressure.

(Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(OAll)-MCA (compound 57))

HD-Glu(OAll)-MCA (compound 28) (90 mg, 0.26 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was dissolved in CHCl ₃ (60 mL), washed with water, 1 mol/L HCl, saturated NaHCO ₃ aqueous solution, and then saturated NaCl water, dried over Na ₂ SO ₄ , filtered, and concentrated to dryness under reduced pressure. The residue was suspended in CHCl ₃ , and n-Hex. was added under stirring and stirred at room temperature. The suspension was filtered and dried (60°C, reduced pressure) to obtain 209 mg of a white solid. It was then dissolved in 5% MeOH/CHCl ₃ and purified by silica gel column chromatography (CHCl ₃ to 10% MeOH/CHCl ₃ ). The mixed fraction of Rf = 0.66 and 0.62 (10% MeOH/CHCl ₃ ) was concentrated to dryness under reduced pressure, and the residue was washed with n-Hex./CHCl ₃ and dried (60°C, reduced pressure) to give compound 57 (144 mg, 63.4%) as a white solid.

From the ¹ H-NMR spectrum, it was estimated to be a mixture of five isomers, with the estimated abundance ratio being 5: 17: 6: 50: 22. In LCMS, it was observed as two retention times.
^1H -NMR (400 MHz, DMSO-d ₆ ) δppm: 10.50, 10.46, 10.41, 10,36, 10,25 (each s, 1H, Coumarin C7-NH; D ₂ O exchange), 8.65-8.10 (m, 2H, NH; D ₂ O exchange [8.61 (d, J = 7.8 Hz), 8.50 (d, J = 7.8 Hz), 8.43 (d, J = 7.3 Hz), 8.35 (d, J = 7.3 Hz), 8.30 (d, J = 7.8 Hz), 8.28 (d, J = 7.8 Hz), 8.15 (d, J = 8.5 Hz)]), 7.84-7.47 (m, 4H, Coumarin C8-H [7.82 (d, J = 2.1 Hz,), 7.79 (d, J = 2.1 Hz), 7.77 (d, J = 2.1 Hz), 7.75 (d, J = 2.1 Hz)], Coumarin C5-H [7.724 (d, J = 8.7 Hz,), 7.718 (d, J = 8.7 Hz), 7.69 (d, J = 8.7 Hz)], 7.63 (d, J = 6.9 Hz, NH; D ₂ O exchange), Coumarin C6-H [7.55 (dd, J = 2.1, 8.7 Hz), 7.53 (dd, J = 2.1, 8.7 Hz), 7.50 (dd, J = 2.1, 8.7 Hz)]), 7.46-7.20 (m, 10H, _OCH2Ph , benzylidene-Ph), 6.28 (d, 1H, J = 1,1 Hz, coumarin C3-H), 5.95-5.82 (m, 1H, _OCH2 -CH= _CH2 ), 5.71, 5.70, 5.69 (each s, 1H, benzylidene-CH), 5.32-5.23 (m, 1H, OCH2a _- CH= _CH2 ), 5.22-5.15 (m, 1H, OCH2b-CH _{= CH2} ), 4.92-4.83 (m, 1H, MurNAc C1-βH [4.89 (d, J = 3.7 Hz), 4.87 (d, J = 3.7 Hz), 4.86 (d, J = 3.7 Hz)]), 4.74-4.62 (m, 1H, Ph CH ₂ a [4.71 (d, Jgem = 12.6 Hz,), 4.69 (d, Jgem = 12.6) Glu-βCH ₂ , Coumarin C4- CH ₃ [2.40(d, J = 1,1 Hz), 2.37 (d, J = 1,1 Hz)]), 2.19-1.75 (m, 5H, Glu-γCH ₂ , NHCO CH ₃ [1.81 (s), 1.80 (s)]), 1.31-1.18 (m, 6H, Ala- CH ₃ or MurNAc- CH ₃ [1.27 (d, J = 7,1 Hz), 1.26 (d, J = 7,1 Hz), 1.22 (d, J = 6.6 Hz), 1.21 (d, J = 6.6 Hz)]).
LCMS m/z: 869.6 [M+H] ⁺ . (Exact Mass: 868.35)
Purity (LCMS; 326 nm): 91.67% (52.98%, 38.69%).

(Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(MCA)-OAll (compound 54))

HD-Glu(MCA)-OAll (compound 27) (325 mg, 0.94 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was dissolved in CHCl ₃ (200 mL), washed with water, 1 mol/L HCl, saturated NaHCO ₃ aqueous solution, and then saturated NaCl water, dried over Na ₂ SO ₄ , filtered, and concentrated to dryness under reduced pressure. The residue was suspended in CHCl ₃ (20 mL), and n-Hex. (40 mL) was added under stirring and stirred at room temperature. The suspension was filtered, washed with n-Hex. (40 mL)/CHCl ₃ (10 mL), and dried (50°C, reduced pressure) to obtain compound 54 (741 mg, 90.4%) as a pale yellow solid. From the ¹ H-NMR spectrum, it was estimated to be a mixture of two isomers (abundance ratio about 3:1).
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.37 (s, 0.25H, Coumarin C7-NH; D ₂ O exchange), 10.35 (s, 0.75H, Coumarin C7-NH; D ₂ O exchange), 8.63 (d, 0.25H, J = 7.6 Hz, NH; D ₂ O exchange), 8.51 (d, 0.75H, J = 7.6 Hz, NH; D ₂ O exchange), 8.40 (d, 0.25H, J = 8.0 Hz, NH; D ₂ O exchange), 8.16 (d, 0.75H, J = 8.0 Hz, NH; D ₂ O exchange), 7.75 (d, 0.25H, J = 2.1 Hz, coumarin C8-H), 7.74 (d, 0.75H, J = 2.1 Hz, coumarin C8-H), 7.70 (d, 0.75H, J = 8.7 Hz, coumarin C5-H), 7.695 (d, 0.25H, J = 8.7 Hz, coumarin C5-H), 7.65 (d, 0.25H, J = 7.8 Hz, NH; D ₂ O exchange), 7.56 (d, 0.75H, J = 7.8 Hz, NH; D ₂ O exchange), 7.50-7.24 (m, 11H, OCH ₂ Ph, benzylidene-Ph, coumarin C6-H), 6.26 (d, 1H, J = 1,1 Hz, Coumarin C3-H), 5.95-5.82 (m, 1H, O CH ₂ -CH= CH ₂ ), 5.701 (s, 0.25H, benzylidene-CH), 5.700 (s, 0.75H, benzylidene-CH), 5.31 (ddt, 0.25H, J = 1.6, 3.2, 17.2 Hz, O CH ₂ a -CH= CH ₂ ), 5.29 (ddt, 0.75H, J = 1.6, 3.2, 17.2 Hz, O CH ₂ a -CH= CH ₂ ), 5.23-5.17 (m, 1H), 4.89 (d, 0.25H, J = 3.7 Hz, MurNAc C1-βH), 4.87 (d, 0.75H, J = 3.7 Hz, MurNAc C1-βH), 4.70 (d, 0.75H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.69 (d, 0.25H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.63-4.53 (m, 2H), 4.51 (d, 0.75H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.47 (d, 0.25 H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.45-4.12 (m, 4H), 4.03-3.91 (m, 1H), 3.84-3.61(m, 4H), 2.46 (t, 2H, J = 7.3Hz, Glu-βCH ₂ ), 2.394 (d, 0.75H, J = 1,1 Hz, Coumarin C4- CH ₃ ), 2.391 (d, 2.25H, J = 1,1 Hz, Coumarin C4- CH ₃ ), 2.21 (m, 1H, Glu-γCH ₂ a), 2.02-1.84 (m, 1H, Glu-γCH ₂ b), 1.81 (s, 0.75H, NHCO CH ₃ ), 1.79 (s, 2.25H, NHCO CH ₃ ), 1.27 (d, 0.75H, J = 7.1 Hz, Ala- CH ₃ or MurNAc- _CH3 ), 1.25 (d, 2.25H, J = 7.1 Hz, Ala- CH ₃ or MurNAc- CH ₃ ), 1.24 (d, 0.75H, J = 6.6 Hz, Ala- CH ₃ or MurNAc- CH ₃ ), 1.21 (d, 2.25H, J = 6.6 Hz, Ala- CH ₃ or MurNAc- CH ₃ ).
LCMS m/z: 869.82 [M+H] ⁺ . (Exact Mass: 868.35)
Purity (LCMS): 89.94% (62.74% + 27.20%)

(Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-MCA (compound 58))

Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-OH (compound 7) (750 mg, 1.38 mmol) was dissolved in a mixture _of dehydrated _CH2Cl2 (30 mL) and dehydrated DMF (2 mL), and _HOBt.H2O (254 mg, 1.66 mmol, 1.2 eq.) and EDCI (318 mg, 1.66 mmol, 1.2 eq.) were added and stirred at room temperature for 30 min. Then, DIEA (357 mg, 2.76 mmol, 2.0 eq.) and HD-isoGln-MCA (compound 30) (500 mg, 1.65 mmol, 1.2 eq.) were added to the reaction mixture and stirred for 2 h. The reaction mixture was diluted with CHCl ₃ , washed with water, saturated aqueous NaHCO ₃ , and saturated aqueous NaCl, then dried over Na ₂ SO ₄ , filtered, and concentrated to dryness under reduced pressure. The residue was dissolved in a 20% CHCl ₃ /MeOH mixture, silica gel (FL-100D, 20 g) was added, concentrated to dryness under reduced pressure, purified by silica gel column chromatography (5% to 10% MeOH/CHCl ₃ ), and dried (50°C, reduced pressure) to obtain compound 58 (910 mg, 79.8%) as a white solid. From the ¹ H-NMR spectrum and LCMS spectrum, it was estimated to be a mixture of two isomers (abundance ratio about 3:7).
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 10.37-10.32 (s-like, 1H, Coumarin C7-NH; _D2O exchange), 8.39-8.11 (m, 2H, NH; _D2O exchange), 7.49 (d, 1H, J = 2.1 Hz), 7.73-7.54 (m, 2H; 1H- D ₂ O exchange), 7.50-7.25 (m, 12H; 1H- D ₂ O exchange), 7.16-7.09 (br-like, 1H, NH; D ₂ O exchange), 6.26 (d, 1H, J = 1.1 Hz), 5.71 (s, 1H), 4.92(d, 0.7H, J = 3.7 Hz), 4.86 (d, 0.3H, J = 3.7Hz), 4.71 (d, 0.3H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.70 (d, 0.7H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.52 (d, 0.3H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.50 (d, 0.7H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.42-4.12 (m, 4H), 4.06-3.978 (m, 0.3H), 3.975-3.89 (m, 0.7H), 3.82-3.63(m, 4H), 2.46-2.35 (m, 5H, CH ₂ & Coumarin C4- CH ₃ ), 2.12-1.99 (m, 1H), 1.95-1.81 (m, 1H), 1.84 (s, 0.7H, NHCO CH ₃ ), 1.81 (s, 0.3H, NHCO CH ₃ ), 1.30-1.19 (m, 6H, _CH3X2 ).
^* 1.253 (d, 2.1H, J = 6.6 Hz; CH ₃ ), 1.246 (d, 2.1H, J = 6.6 Hz; CH ₃ ), 1.28-1.23 (0.9H; CH ₃ ), 1.21 (d, 0.9H, J = 6.6 Hz; CH ₃ ).
LCMS m/z: 828.8 [M+H] ⁺ . (Exact Mass: 827.34)
Purity (LCMS): 99.81% (29.36%, 70.45%).

(Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-pNA (compound 56))

HD-isoGln-pNA (compound 31) (400 mg, 1.50 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was dissolved in CHCl ₃ (180 mL), and water (80 mL) was added to separate the layers. The white solid precipitated in the aqueous layer was collected by filtration, dissolved in 20% MeOH/CHCl ₃ , dried over Na ₂ SO ₄ , filtered, and concentrated to dryness under reduced pressure to obtain a pale yellow viscous oily residue. The residue was treated with n-Hex. (25 mL)/CHCl ₃ (25 mL), and the resulting white gel was collected by filtration and dried (50°C, reduced pressure) to obtain compound 56 (622 mg, 52.4%) as a pale yellow solid. From the ¹ H-NMR spectrum and LCMS spectrum, it was estimated to be a mixture of two isomers (abundance ratio approximately 5.7:1).
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.53 (s, 1H, pNA-NH; D ₂ O exchange), 8.36 (d, 0.15H, J = 8.5 Hz, NH; D ₂ O exchange), 8.24-8.17 (m, 3H [8.21 (d, 2H, J = 9.4 Hz, pNA C3,5-H), 8.20 (d, 0.85H, J = 8.5 Hz, NH; D ₂ O exchange), 0.15H, NH; D ₂ O exchange]), 8.14 (d, 0.85H, J = 8.5 Hz, NH; D ₂ O exchange), 7.82 (d, 2H, J = 9.4 Hz, pNA C2,6-H), 7.69 (d, 0.15H, J = 7.1 Hz, NH; _D2O exchange), 7.56 (d, 0.85H, J = 7.1 Hz, NH; _D2O exchange), 7.47-7.25 (m, 11H, benzylidene-Ph, benzyl-Ph, isoGln- _NH2a ; _D2O exchange), 7.13 (br-s, 0.15H, _isoGln -NH2b; _D2O exchange), 7.11 (br-s, 0.85H, isoGln- _NH2b ; D2O exchange), 5.71 (s, 1H, benzylidene-CH ₎ , 4.91 (d, 0.15H, J = 3.7 Hz, MurNAc C1-βH), 4.86 (d, 0.85H, J = 3.7 Hz, MurNAc C1-βH), 4.71 (d, 0.85H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.70 (d, 0.15H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.51 (d, 0.85H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.49 (d, 0.15H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.40-4.12 (m, 4H), 4.06-3.89 (m, 1H), 3.83-3.63(m, 4H), 2.44(t, 0.15H, J = 7.8 Hz, isoGln-βCH ₂ ), 2.41(t, 0.85H, J = 7.8 Hz, isoGln-βCH ₂ ), 2.12-2.01(m, 1H, isoGln-γCH ₂ a), 1.86-1.74(m, 4H [1.86-1.74 (m, 1H, isoGln-γCH ₂ b), 1.84 (s, 0.45H, NHCO CH ₃ ), 1.80 (s, 2.55H, NHCO CH ₃ ]), 1.29-1.16 (m, 6H [1,244 (d, 0.45H, J = 6.9 Hz, Ala- CH ₃ or MurNAc- CH ₃ ), 1.237 (d, 2.55H, J = 6.9 Hz, Ala- CH ₃ or MurNAc- CH ₃ ), 1.20 (d, 2.55H, J = 6.6 Hz, Ala- CH ₃ or MurNAc- CH ₃ ]).
LCMS m/z: 791.8 [M+H] ⁺ . (Exact Mass: 790.32)
Purity (LCMS): 97.05% (79.14%, 17.91%).

(Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-D-isoGln-pNA (compound 59))

HD-isoGln-pNA (compound 31) (312 mg, 1.17 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. A pale yellow syrup-like residue was obtained. The residue was suspended in CHCl ₃ (10 mL), and n-Hex. (40 mL) was added and stirred. The suspension was filtered, washed with water, and dried (50°C, reduced pressure) to obtain compound 59 (973 mg, quant.) as a white solid. From ^{the 1} H-NMR spectrum and LCMS spectrum, it was estimated to be a mixture of two isomers (abundance ratio approximately 3:1).
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.53 (s, 1H, pNA-NH; D ₂ O exchange), 8.37 (d, 0.75H, J = 7.6 Hz, CONH; D ₂ O exchange), 8.24-8.11 (m, 3.25H [8.20 (d, 2H, J = 9.4 Hz, pNA C3,5-H), 8.15 (d, 0.25H, J = 8.2 Hz, CONH; D ₂ O exchange), 0.25H, CONH; D ₂ O exchange]), 8.14 (d, 0.75H, J = 8.2 Hz, CONH; D ₂ O exchange), 7.82 (d, 2H, J = 9.4 Hz, pNA C2,6-H), 7.70 (d, 0.75H, J = 7.3 Hz, CONH; _D2O exchange), 7.69 (d, 0.25H, J = 7.4 Hz, CONH; _D2O exchange), 7.47-7.25 (m, 11H, benzylidene-Ph, benzyl-Ph, isoGln- _CONH2a ; _D2O exchange), 7.13 (br-s, 0.75H, isoGln- _CONH2b ; _D2O exchange), 7.11 (br-s, 0.25H, isoGln- _CONH2b ; _D2O exchange), 5.71 (s, 1H, benzylidene-CH), 4.92 (d, 0.75H, J = 3.7 Hz, MurNAc C1-βH), 4.86 (d, 0.25H, J = 3.7 Hz, MurNAc C1-βH), 4.71 (d, 0.25H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.70 (d, 0.75H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.51 (d, 0.25H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.50 (d, 0.75H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.41-4.11 (m, 4H), 4.05-3.86 (m, 1H), 3.84-3.62 (m, 4H), 2.44 (t, 0.75H, J = 8.0 Hz, isoGln-βCH ₂ ), 2.41(t, 0.25H, J = 8.0 Hz, isoGln-βCH ₂ ), 2.12-1.96(m, 1H, isoGln-γCH ₂ a), 1.94-1.77(m, 4H [1.94-1.77 (m, 1H, isoGln-γCH ₂ b), 1.84 (s, 2.25H, NHCO CH ₃ ), 1.81 (s, 0.75H, NHCO CH ₃ ]), 1.28-1.18 (m, 6H [1.25 (d, 2.25H, J = 7.1 Hz, Ala- CH ₃ or MurNAc- CH ₃ ), 1.24 (d, 2.25H, J = 6.6 Hz, Ala- CH ₃ or MurNAc- CH ₃ ), 1.20 (d, 0.75H, J = 6.6 Hz, Ala- CH ₃ or MurNAc- CH ₃ ]).
LCMS m/z: 791.8 [M+H] ⁺ . (Exact Mass: 790.32)
Purity (LCMS): 81.26% (20.68% + 60.58%)

Synthesis of HL-Ala-D-isoGln-MCA・HCl
(Synthesis of Fmoc-L-Ala-D-isoGln-MCA (Compound 39))

Fmoc-L-Ala-OH (compound 33) (87 mg, 0.28 mmol) was dissolved in a mixture _of dehydrated _CH2Cl2 (6 mL) and dehydrated DMF (0.4 mL), and _HOBt.H2O (51 mg, 0.33 mmol, 1.2 eq.) and EDCI (63 mg, 0.33 mmol, 1.2 eq.) were added and stirred at room temperature for 30 minutes. DIEA (72 mg, 0.56 mmol, 2.0 eq.) and HD-isoGln-MCA (compound 30) (100 mg, 0.33 mmol, 1.2 eq.) were then added to the reaction mixture and stirred for 3 hours. The reaction mixture was concentrated to dryness under reduced pressure. The residue was dissolved in 20% CHCl ₃ /MeOH, silica gel (FL-100D, 4 g) was added, and the mixture was concentrated to dryness under reduced pressure and purified by silica gel column chromatography (5% to 10% MeOH/CHCl ₃ ). The target fraction was collected and concentrated to dryness under reduced pressure. The residue was washed with n-Hex./CHCl ₃ and dried (50°C, reduced pressure) to give compound 39 (98 mg, 58.7%) as a white solid.
^1H -NMR (400 MHz, DMSO-d ₆ ) δppm: 10.36 (s, 1H, Coumarin C7-NH; D ₂ O exchange), 8.09 (d, 1H, J = 8.2 Hz, NH; D ₂ O exchange), 7.88 (d, 2H, J = 7.6 Hz), 7.78-7.65 (m, 4H), 7.61 (d, 1H, J = 7.1 Hz, NH; D ₂ O exchange), 7.50-7.37 (m, 3H), 7.36-7.27 (m, 3H; 1H- D ₂ O exchange), 7.15 (s, 1H, NH; D ₂ O exchange), 6.25 (d, 1H, J = 1.1 Hz, Coumarin C3-H), 4.31-4.17 (m, 4H), 4.13-4.04 (m, 1H), 2.43-2.36 (m, 2H), 2.39 (d, 3H, J = 1.1 Hz, Coumarin C4- CH ₃ ), 2.15-1.99 (m, 1H), 1.93-1.75 (m, 1H), 1.23 (d, 3H, J = 7.1 Hz, Ala- CH ₃ ).
LCMS m/z: 597.6 [M+H] ⁺ . (Exact Mass: 596.23)
Purity (LCMS): 98.28%

<Benzyl-4,6-O-benzylidene-α-MurNAc (General Method)>
Benzyl-4,6-O-benzylidene-α-MurNAc (compound 4) (1.5 eq.) was dissolved in dehydrated DMF (75 mL/g), and N-methylmorpholine (4.5 eq.) and HBTU (3.0 eq.) were added under an argon gas atmosphere and stirred at room temperature for 15 minutes. Then, HL-Ala-D-Glu, HL-Ala-D-Gln, or HL-Ala-D-isoGln derivatives (1.0 eq.) were added and stirred for 24 hours, and the reaction mixture was concentrated to dryness under reduced pressure.

(Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Gln-MCA (compound 55))

HL-Ala-D-Gln-MCA (compound 44) (186 mg, 0.50 mmol) was used in the reaction according to the general method, and the reaction solution was concentrated to dryness under reduced pressure. The residue was suspended in AcOEt and washed with water (80 mL). The AcOEt layer (suspension) was separated and washed with 1 mol/L HCl (100 mL), saturated NaHCO ₃ aqueous solution (100 mL), and then saturated NaCl water (120 mL). The AcOEt layer (suspension) was separated and concentrated to dryness under reduced pressure. The residue was washed with n-Hex. (25 mL)/CHCl ₃ (20 mL), then water, and dried (50°C, reduced pressure) to obtain compound 55 (412 mg, quant.) as a pale yellow to white solid. From the ¹ H-NMR spectrum, it was estimated to be a mixture of two isomers (abundance ratio about 5:1).
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.35 (s, 0.85H, Coumarin C7-NH; D ₂ O exchange), 10.31 (s, 0.15H, Coumarin C7-NH; D ₂ O exchange), 8.49 (d, 1H, J = 7.8 Hz, NH; D ₂ O exchange), 8.17 (d, 0.15H, J = 8.2 Hz, NH; D ₂ O exchange), 8.15 (d, 0.85H, J = 8.2 Hz, NH; D ₂ O exchange), 7.83 (d, 0.15H, J = 2.1 Hz, Coumarin C8-H), 7.79 (d, 0.85H, J = 2.1 Hz, Coumarin C8-H), 7.74 (d, 0.15H, J = 8.7 Hz ; Coumarin C5-H), 7.72 (d, 0.85H, J = 8.7 Hz ; Coumarin C5-H), 7.60 (d, 1H, J = 7.1 Hz, NH; D ₂ O exchange), 7.55 (dd, 1H, J = 2.1, 8.7 Hz, Coumarin C6-H), 7.44-7.26 (m, 11H, Ph X 2, Gln- CONH ₂ a; D ₂ O exchange), 6.78 (s, 1H, Gln- CONH ₂ b; D ₂ O exchange), 6.28 (d, 1H, J = 1.1 Hz, Coumarin C3-H), 5.69 (s, 0.85H, benzylidene-CH), 5.67 (s, 0.15H, benzylidene-CH), 4.87 (d, 0.15H, J = 3.7 Hz, MurNac C1-βH), 4.86 (d, 0.85H, J = 3.7 Hz, MurNAc C1-βH), 4.71 (d, 0.85H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.69 (d, 0.15H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.51 (d, 0.85H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.48 (d, 0.15H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.43-4.26 (m, 2H), 4.25-4.11 (m, 2H,), 4.06-3.96 (m, 1H), 3.82-3.64 (m, 4H), 2.40 (d, 3H, J = 1.1 Hz, Coumarin C4- CH ₃ ), 2.18-1.95 (m, 3H, Gln-βCH ₂ , Gln-γCH ₂ a), 1.87-1.76 (m, 1H, Gln-γCH ₂ b), 1.84 (s, 0.45H, NHCO CH ₃ ), 1.80 (s, 2.55H, NHCO CH ₃ ), 1.27(d, 2.55H, J = 6.9 Hz, CH ₃ ), 1.23 (d, 0.45H, J = 6.9 Hz, CH ₃ ), 1.21 (d, 2.55H, J = 6.9 Hz, CH ₃ ), 1.19 (d, 0.45H, J = 6.9 Hz, CH ₃ ).
LCMS m/z: 828.7 [M+H] ⁺ . (Exact Mass: 827.34)
Purity (LCMS): 98.53% (86.66% + 11.87%)

(Synthesis of benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-pNA (compound 56))

Benzyl-4,6-O-benzylidene-α-MurNAc (compound 4) (226 mg, 0.48 mmol) and HL-Ala-D-isoGln-pNA (compound 46) (210 mg, 0.62 mmol, 1.3eq.) were used to react according to the general method. Water (5 mL) was added to the reaction mixture (suspension), and AcOEt was added and the layers were separated. The AcOEt layer (suspension) was separated and washed with 1 mol/L HCl, saturated NaHCO ₃ aqueous solution, and then water. The AcOEt layer (suspension) was separated and concentrated to dryness under reduced pressure. The residue was suspended in n-Hex. (15 mL)/CHCl ₃ (15 mL), washed, and dried (60°C, reduced pressure) to obtain compound 56 (399 mg, quant.) as a white solid. From ¹ H-NMR spectrum and LCMS, it was estimated to be a mixture of two isomers (abundance ratio: about 5.7:1).
^1H -NMR (400 MHz, DMSO-d ₆ ) δppm: 10.57 (s, 1H, pNA-NH; D ₂ O exchange), 8.30-8.18 (br, 1H, NH; D ₂ O exchange), 8.20 (d, 2H, J = 9.4 Hz, pNA C3,5-H), 8.15 (br-d, 1H, J = 8.2 Hz, NH; D ₂ O exchange), 7.82 (d, 2H, J = 9.4 Hz, pNA C2,6-H), 7.75 (br-d, 1H, J = 6.2 Hz, NH; D ₂ O exchange), 7.47-7.26 (m, 11H), Benzylidene-Ph, Benzyl-Ph, isoGln- NH ₂ a; D ₂ O exchange), 7.10 (br-s, 1H, isoGln- NH ₂ b; D ₂ O exchange), 5.71 (s, 0.85H, benzylidene-CH), 5.70 (s, 0.15H, benzylidene-CH), 4.88 (d, 0.15H, J = 3.7 Hz, MurNAc C1-βH), 4.86 (d, 0.85H, J = 3.7 Hz, MurNAc C1-βH), 4.71 (d, 1H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.51 (d, 1H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.26-4.12 (m, 4H), 4.06-3.95 (m, 1H), 3.84-3.66 (m, 4H), 2.41 (t, 2H, J = 7.8 Hz, isoGln-βCH ₂ ), 2.13-2.00 (m, 1H, isoGln-γCH ₂ a), 1.87- 1.75 (m, 4H, isoGln-γCH ₂ b [1.81 (s, 0.45H, NHCO CH ₃ ), 1.80 (s, 2.55H, NHCO CH ₃ )]), 1.24 (d, 3H, J = 6.9 Hz, L-Ala- CH ₃ or MurNAc- CH ₃ ), 1.20 (d, 3H, J = 6.6 Hz, L-Ala- CH ₃ or MurNAc- CH ₃ ).
LCMS m/z: 791.8 [M+H] ⁺ . (Exact Mass: 790.32)
Purity (LCMS): 98.61% (80.66% + 17.95%)

<Debenzylidene synthesis (general method)>
The benzylidene derivative was suspended in 75% AcOH (75-170 mL/g) and stirred for 5-23 hours at 50-60° C. The disappearance of the raw materials was confirmed by TLC (10% MeOH/CHCl ₃ ), and the reaction solution was concentrated to dryness under reduced pressure to obtain a colorless, starch-syrup-like residue.

(Synthesis of benzyl-α-MurNAc-L-Ala-D-Glu(OAll)-MCA (compound 61))

Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(OAll)-MCA (compound 57) (130 mg, 0.15 mmol) was used in the reaction according to the general method, and the mixture was concentrated to dryness under reduced pressure. The residue was crystallized from n-Hex./AcOEt (3:1) and dried (60°C, reduced pressure) to obtain compound 61 (115 mg, 98.3%) as a white solid. ^1H -NMR spectrum and LCMS suggested that it was a mixture of five isomers. The abundance ratio was estimated to be 2: 17: 7: 50: 24.
^1H -NMR (400 MHz, DMSO-d ₆ ) δppm: 10.51, 10.46, 10.42, 10,35, 10,32 (each s, 1H, Coumarin C7-NH; D ₂ O exchange), 8.59-8.06 (m, 2H, NH; D ₂ O exchange [8.55 (d, J = 8.0 Hz), 8.50 (d, J = 8.0 Hz), 8.83 (d, J = 7.3 Hz), 8.31 (d, J = 7.3 Hz), 8.30 (d, J = 7.3 Hz), 8.28 (d, J = 7.3 Hz), 8.10 (d, J = 8.0 Hz)]), 7.82-7.47 (m, 4H, Coumarin C8-H [7.80 (d, J = 2.1 Hz)], Coumarin C5-H [7.73 (d, J = 8.7 Hz)], 7.63 (d, J = 6.9 Hz, NH; D ₂ O exchange), Coumarin C6-H [7.56 (dd, J = 2.1, _{D 2 O ​} exchange [5.38(d, J = 6.9 Hz), 5.37 (d, J = 6.9 Hz), 5.35 (d, J = 6.9 Hz), 5.28 (d, J = 6.9 Hz)], O CH ₂ a -CH= CH ₂ [5.32-5.24 (m, 1H)], O CH ₂ b -CH= CH ₂ [5.22-5.16 (m, 1H)]), 4.93-4.22(m, 9H, MurNAc C6-OH; D ₂ O exchange, MurNAc C1-βH [4.82(d, J = 3.4 Hz), 4.79(d, J = 3.4 Hz), 4.74(d, J = 3.4 Hz)], Ph CH ₂ a [4.67(d, Jgem = 12.6 Hz)]), 3.88-3.59 (m, 2H), 3.57-3.40 (m, 3H), 3.39-3.26 (m, 1H), 2.48-2.38 (m, 5H, Glu-βCH ₂ , Coumarin C4- CH ₃ [2.40 (d, J = 1,1 Hz)]), 2.19-2.02 (m, 1H, Glu-γCH ₂ a), 1.99-1.75 (m, 1H, Glu-γCH ₂ b), 1.80, 1.794, 1.786 (each s, 3H, NHCO CH ₃ ), 1.34-1.20 (m, 6H, Ala- CH ₃ or MurNAc- CH ₃ [1.27 (d, J = 7,1 Hz), 1.26 (d, J = 6.6 Hz)]).
LCMS m/z: 781.6 [M+H] ⁺ . (Exact Mass: 780.32)
Purity (LCMS; 326 nm): 89.08% (6.61%, 44.59%, 15.07%, 21.34%, 1.47%).

(Synthesis of benzyl-α-MurNAc-L-Ala-D-Glu(MCA)-OAll (compound 62))

Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Glu(MCA)-OAll (compound 54) (500 mg, 0.58 mmol) was used in the reaction according to the general method, and the mixture was concentrated to dryness under reduced pressure. The residue was crystallized from n-Hex./AcOEt (1:1) and dried (50°C, reduced pressure) to obtain compound 62 (440 mg, 98.0%) as a pale ochre solid. From the ^1H -NMR spectrum, it was estimated to be a mixture of two isomers (abundance ratio approximately 3:1).
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.38 (s, 0.25H, Coumarin C7-NH; D ₂ O exchange), 10.36 (s, 0.75H, Coumarin C7-NH; D ₂ O exchange), 8.59 (d, 0.25H, J = 7.6 Hz, NH; D ₂ O exchange), 8.50 (d, 0.75H, J = 7.6 Hz, NH; D ₂ O exchange), 8.36 (d, 0.25H, J = 8.0 Hz, NH; D ₂ O exchange), 8.12 (d, 0.75H, J = 8.0 Hz, NH; D ₂ O exchange), 7.76 (d, 0.25H, J = 2.1 Hz, coumarin C8-H), 7.74 (d, 0.75H, J = 2.1 Hz, coumarin C8-H), 7.71 (d, 0.75H, J = 8.7 Hz, coumarin C5-H), 7.70 (d, 0.25H, J = 8.7 Hz, coumarin C5-H), 7.69 (d, 0.25H, J = 7.8 Hz, NH; D ₂ O exchange), 7.58 (d, 0.75H, J = 7.8 Hz, NH; D ₂ O exchange), 7.47 (dd, 0.75H, J = 2.1, 8.7 Hz, coumarin C6-H), 7.45 (dd, 0.25H, J = 2.1, 8.7 Hz, Coumarin C6-H), 7.40-7.23 (m, 5H, O CH ₂ Ph), 6.26 (d, 1H, J = 1.1 Hz, Coumarin C3-H), 5.96-5.83 (m, 1H, O CH ₂ -CH= CH ₂ ), 5.37 (d, 0.25H, J = 6.9Hz, MurNAc C4-OH; D ₂ O exchange), 5.31 (d, 0.75H, J = 6.9 Hz, MurNAc C4-OH; D ₂ O exchange), 5.34-5.27 (m, 1H, O CH ₂ -CH= CH ₂ a), 5.24-5.18 (m, 1H, O CH ₂ -CH= CH ₂ b), 4.80(d, 0.25H, J = 3.4 Hz, MurNAc C1-βH), 4.75 (d, 0.75H, J = 3.4 Hz, MurNAc C1-βH), 4.67 (d, 0.75H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.64 (d, 0.25H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.63-4.55(m, 3H, [1H-MurNAc C6-OH; D ₂ O exchange]), 4.52-4.26(m, 4H, [4.43(d, 0.75H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.41(d, 0.25H, Jgem = 12.6 Hz, Ph- CH ₂ b)]), 3.85-3.60 (m, 2H), 3.57-3.42 (m, 3H), 3.40-3.26 (m, 1H), 2.47 (t, 2H, J = 7.6 Hz, Glu-βCH ₂ ), 2.40 (d, 3H, J = 1.1 Hz, Coumarin C4- CH ₃ ), 2.21-2.06 (m, 1H, Gln-γCH ₂ a), 2.00-1.83 (m, 1H, Gln-γCH ₂ b), 1.80 (s, 0.75H, NHCO CH ₃ ), 1.73 (s, 2.25H, NHCO CH ₃ ), 1.30-1.22(m, 6H [1.26(d, 2.25H, J = 6.6 Hz, Ala- CH ₃ or MurNAc- CH ₃ ), 1.25(d, 2.25H, J = 6.6 Hz, Ala- CH ₃ or MurNAc- CH ₃ )).
LCMS m/z: 781.8 [M+H] ⁺ . (Exact Mass: 780.32)
Purity (LCMS): 84.68% (60.31% + 24.37%)

(Synthesis of benzyl-α-MurNAc-L-Ala-D-Gln-MCA (compound 60))

Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-Gln-MCA (compound 55) (384 mg, 0.46 mmol) was used in the reaction according to the general method, and the mixture was concentrated to dryness under reduced pressure. The residue was crystallized from n-Hex./CHCl ₃ (1:1) and dried (50°C, reduced pressure) to obtain compound 60 (327 mg, 95.3%) as a white solid. From the ^1H -NMR spectrum, it was estimated to be a mixture of two isomers (abundance ratio approximately 4:1).
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 10.35 (s, 0.8H, coumarin C7-NH; _D2O exchange), 10.31 (s, 0.2H, coumarin C7-NH; _D2O exchange), 8.49 (d, 1H, J = 8.0 Hz, NH; _D2O exchange), 8.28 (d, 0.2H, J = 8.2 Hz, NH; _D2O exchange), 8.11 (d, 0.8H, J = 8.2 Hz, NH; _D2O exchange), 7.85 (d, 0.2H, J = 1.8 Hz, coumarin C8-H), 7.81 (d, 0.8H, J = 1.8 Hz, coumarin C8-H), 7.75 (d, 0.2H, J = 8.7 Hz, coumarin C5-H), 7.73 (d, 0.8H, J = 8.7 Hz, coumarin C5-H), 7.61 (d, 1H, J = 7.1 Hz, NH; _D2O exchange), 7.57 (dd, 1H, J = 1.8, 8.7 Hz, coumarin C6-H), 7.40-7.24 ( _m , 6H, Ph, _CONH2a ; _D2O exchange), 6.79 (s, 1H, CONH2b; _D2O exchange), 6.28 (d, 1H, J = 1.1 Hz; coumarin C3-H), 5.52 (d, 0.2H, J = 7.1 Hz, MurNAc C4-OH; D ₂ O exchange), 5.29 (d, 0.8H, J = 7.1 Hz, MurNAc C4-OH; D ₂ O exchange), 4.76 (d, 0.2H, J = 3.4 Hz), 4.74 (d, 0.8H, J = 3.4 Hz), 4.66 (d, 1H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.62 (t, 0.2H, J = 6.0 Hz, MurNAc C6-OH; D ₂ O exchange), 4.59 (t, 0.8H, J = 6.0 Hz, MurNAc C6-OH; D ₂ O exchange), 4.47-4.23 (m, 3H), 4.442(d, 0.2H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.436 (d, 0.8H, Jgem = 12.6 Hz, Ph- CH ₂ b), 3.86-3.78 (m, 1H), 3.69-3.60 (m, 1H), 3.86-3.44 (m, 2H), 3.35-3.26 (m, 1H), 2.40 (d, 3H, J = 1.1 Hz, Coumarin C4- CH ₃ ), 2.18-2.10 (m, 2H, Gln-βCH ₂ ), 2.07-1.97 (m, 1H, Gln-γCH ₂ a), 1.88-1.78(m, 1H, Gln- _γCH2b ), 1.82 (s, 0.6H, NHCO CH ₃ ), 1.79 (s, 2.4H, NHCO CH ₃ ), 1.27 (d, 3H, J = 6.9 Hz, CH ₃ ), 1.26 (d, 3H, J = 6.6 Hz, CH ₃ ).
LCMS m/z: 740.7 [M+H] ⁺ . (Exact Mass: 739.31)
Purity (LCMS): 79.34%

(Synthesis of benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (compound 63))

Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-MCA (compound 58) (500 mg, 0.60 mmol) was used in the reaction according to the general method, and the reaction mixture was concentrated to dryness under reduced pressure. The residue was crystallized from n-Hex. (10 mL)/CHCl ₃ (10 mL), dissolved in 20% MeOH/CHCl ₃ , silica gel (FL 100D, 10 g) was added, and the mixture was concentrated to dryness under reduced pressure. The mixture was purified by silica gel column chromatography (5% to 12% MeOH/CHCl ₃ ) and dried (50 °C, reduced pressure) to give compound 63 (63A: Rf = 0.25 [10% MeOH/CHCl ₃ ] 198 mg, 63B: Rf = 0.19 [10% MeOH/CHCl ₃ ] 54 mg, 86 mg as a mixture) as a white solid (total yield 338 mg, 75.6%).

(Compound 63A)
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 10.35 (s, 1H, coumarin C7-NH; _D2O exchange), 8.33 (d, 1H, J = 7.1 Hz, isoGln-NH; _D2O exchange), 8.10 (d, 1H, J = 8.0 Hz, NH-Ac; _D2O exchange), 7.79 (d, 1H, J = 7.1 Hz, Ala-NH; _D2O exchange), 7.76 (d, 1H, J = 2.1 Hz, coumarin C8-H), 7.69 (d, 1H, J = 8.7 Hz, coumarin C5-H), 7.45 (dd, 1H, J = 2.1, 8.7 Hz, Coumarin C6-H), 7.38-7.33 (m, 5H, Ph-4H, CONH ₂ a; D ₂ O exchange), 7.31-7.24 (m, 1H, Ph-1H), 7.12 (s, 1H, CONH ₂ b; D ₂ O exchange), 6.26 (d, 1H, J = 1.1 Hz, Coumarin C3-H), 5.38 (d, 1H, J = 6.9 Hz, MurNAc C4-OH; D ₂ O exchange), 4.83 (d, 1H, J = 3.4 Hz), 4.66 (d, 1H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.60 (t, 1H, J = 6.0 Hz, MurNAc C6-OH; D ₂ O exchange), 4.46 (q, 1H, J = 6.6 Hz, MurNAc-OCH CH ₃ ), 4.42 (d, 1H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.37 (pent., 1H, J = 7.1Hz, Ala-αH), 4.29-4.20 (m, 1H), 3.75-3.60 (m, 2H), 3.57-3.42 (m, 3H), 3.40-3.33 (m, 1H), 2.46-2.37 (m, 2H), 2.39 (d, 3H, J = 1.1 Hz, Coumarin C4- CH ₃ ), 2.12-1.98 (m, 1H), 1.94-1.81 (m, 1H), 1.82 (s, 3H, NHCO CH ₃ ), 1.27 (d, 3H, J = 6.6 Hz, MurNAc- CH ₃ ), 1.26 (d, 3H, J = 7.1 Hz, Ala- CH ₃ ).
LCMS m/z: 740.5 [M+H] ⁺ . (Exact Mass: 739.31)
Purity (LCMS): 99.09%

(Compound 63B)
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.35 (s, 1H, Coumarin C7-NH; D ₂ O exchange), 8.19 (d, 1H, J = 8.2 Hz, isoGln-NH or NH-Ac; D ₂ O exchange), 8.10 (d, 1H, J = 8.2 Hz, isoGln-NH or NH-Ac; D ₂ O exchange), 7.75 (d, 1H, J = 2.1 Hz, Coumarin C8-H), 7.70 (d, 1H, J = 8.7 Hz, Coumarin C5-H), 7.57 (d, 1H, J = 6.9 Hz, Ala-NH; D ₂ O exchange), 7.47 (dd, 1H, J = 2.1, 8.7 Hz, Coumarin C6-H), 7.41-7.32 (m, 5H, Ph-4H, CONH ₂ a; D ₂ O exchange), 7.31-7.25 (m, 1H, Ph-H), 7.10 (s, 1H, CONH ₂ b; D ₂ O exchange), 6.26 (d, 1H, J = 1.1 Hz; Coumarin C3-H), 5.30 (d, 1H, J = 6.9 Hz, MurNAc C4-OH; D ₂ O exchange), 4.74 (d, 1H, J = 3.4 Hz), 4.67 (d, 1H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.60 (t, 1H, J = 6.0 Hz, MurNAc C6-OH; D ₂ O exchange), 4.44 (d, 1H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.35-4.17 (m, 3H), 3.86-3.77 (m, 1H), 3.70-3.61 (m, 1H), 3.57-3.44 (m, 3H), 3.36-3.27 (m, 1H), 2.44-2.35 (m, 2H), 2.39 (d, 3H, J = 1.1 Hz, Coumarin C4- CH ₃ ), 2.13-2.00 (m, 1H), 1.88-1.74 (m, 1H), 1.79 (s, 3H, NHCO CH ₃ ), 1.25 (d, 3H, J = 6.6 Hz, MurNAc- CH ₃ ), 1.24 (d, 3H, J = 7.1 Hz, Ala- CH ₃ ).
LCMS m/z: 740.5 [M+H] ⁺ . (Exact Mass: 739.31)
Purity (LCMS): 99.62%

(Synthesis of benzyl-α-MurNAc-L-Ala-D-isoGln-pNA (compound 64))

Benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-pNA (compound 56) (596 mg, 0.75- mmol) was used in the reaction according to the general method, and the mixture was concentrated to dryness under reduced pressure. The residue was dissolved in 10% MeOH/CHCl ₃ and purified by silica gel column chromatography (5% to 14% MeOH/CHCl ₃ ). The fractions were combined, concentrated to dryness, and dried (50 °C, reduced pressure) to give compound 64 (64A: pale yellow solid Rf = 0.12 [10% MeOH/CHCl ₃ ] 44 mg, 64B: white solid Rf = 0.08 [10% MeOH/CHCl ₃ ] 166 mg, and white solid mixture 222 mg) (total yield 432 mg, 81.5%).

(Compound 64A)
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.54 (s, 1H, pNA-NH; D ₂ O exchange), 8.34 (d, 1H, J = 7.7 Hz, NH; D ₂ O exchange), 8.20 (d, 2H, J = 9.2 Hz, pNA C3,5-H), 8.11 (d, 1H, J = 8.0 Hz, NH; D ₂ O exchange), 7.82 (d, 2H, J = 9.2 Hz, pNA C2,6-H), 7.79 (d, 1H, J = 7.3 Hz, NH; D ₂ O exchange), 7.38-7.24 (m, 6H, benzyl-Ph, CONH ₂ a; D ₂ O exchange), 7.13 (s, 1H, CONH ₂ b; D ₂ O exchange), 5.39 (d, 1H, J = 6.9 Hz, MurNAc C4-OH; D ₂ O exchange), 4.83 (d, 1H, J = 3.4 Hz, MurNAc C1-βH), 4.65 (d, 1H, Jgem = 12.4 Hz, Ph- CH ₂ a), 4.61 (br-t, 1H, J = 5.3 Hz, MurNAc C6-OH; D ₂ O exchange), 4.50-4.33 (m, 3H [4.22 (d, 1H, Jgme = 12.4 Hz, Benzyl- CH ₂ b), 4.36 (t, 1H, J = 7.1 Hz)]), 4.29-4.19 (m, 1H), 3.75-3.60 (m, 2H), 3.57-3.42 (m, 3H), 3.40-3.30 (m, 1H), 2.44 (t, 2H, J = 8.0 Hz, isoGln-βCH ₂ ), 2.11-1.98 (m, 1H, isoGln-γCH ₂ a), 1.94-1.77 (m, 1H, isoGln-γCH ₂ b), 1.81 (s, 3H, NHCO CH ₃ ), 1.26 (d, 3H, J = 6.6 Hz, Ala- CH ₃ or MurNAc- CH ₃ ), 1.25 (d, 3H, J = 6.9 Hz, Ala- CH ₃ or MurNAc- CH ₃ ).
LCMS m/z: 703.7 [M+H] ⁺ . (Exact Mass: 702.29)
Purity (LCMS): 97.63%

(Compound 64B)
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.54 (s, 1H, pNA-NH; D ₂ O exchange), 8.21 (d, 2H, J = 9.2 Hz, pNA C3,5-H), 8.20 (d, 1H, J = 8.0 Hz, NH; D ₂ O exchange), 8.11 (d, 1H, J = 8.2 Hz, NH; D ₂ O exchange), 7.83 (d, 2H, J = 9.2 Hz, pNA C2,6-H), 7.57 (d, 1H, J = 6.9 Hz, NH; D ₂ O exchange), 7.42-7.25 (m, 6H, benzyl-Ph, CONH ₂ a; D ₂ O exchange), 7.11 (s, 1H, CONH ₂ b; D ₂ O exchange), 5.31 (d, 1H, J = 6.9 Hz, MurNAc C4-OH; D ₂ O exchange), 4.74 (d, 1H, J = 3.4 Hz, MurNAc C1-βH), 4.67 (d, 1H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.61 (t, 1H, J = 6.0 Hz, MurNAc C6-OH; D ₂ O exchange), 4.44 (d, 1H, Jgme = 12.6 Hz, Ph- CH ₂ b), 4.35-4.17 (m, 3H), 3.87-3.77 (m, 1H), 3.71-3.60(m, 1H), 3.57-3.44 (m, 3H), 3.36-3.26 (m, 1H), 2.41 (t, 2H, J = 7.8 Hz, isoGln-βCH ₂ ), 2.13-2.00 (m, 1H, isoGln-γCH ₂ a), 1.86-1.73 (m, 1H, isoGln-γCH ₂ b), 1.79 (s, 3H, NHCO CH ₃ ), 1.25 (d, 3H, J = 7.1 Hz, Ala- CH ₃ or MurNAc- CH ₃ ), 1.23 (d, 3H, J = 7.6 Hz, Ala- CH ₃ or MurNAc- CH ₃ ).
LCMS m/z: 703.7 [M+H] ⁺ . (Exact Mass: 702.29)
Purity (LCMS): 99.45%

(Synthesis of benzyl-α-MurNAc-D-Ala-D-isoGln-pNA (compound 65))

Benzyl-4,6-O-benzylidene-α-MurNAc-D-Ala-D-isoGln-pNA (compound 59) (900 mg, 1.14 mmol) was used in the reaction according to the general method, and the mixture was concentrated to dryness under reduced pressure. The residue was dissolved in 10% MeOH/CHCl ₃ and purified by silica gel column chromatography (5% to 15% MeOH/CHCl ₃ ). The fractions were combined, concentrated to dryness, and dried (50 °C, reduced pressure) to give compound 65 (65A: Rf = 0.14 [10% MeOH/CHCl ₃ ] 440 mg, 65B: Rf = 0.09 [10% MeOH/CHCl ₃ ] 72 mg, 109 mg as a mixture) as a white solid (total yield 621 mg, 77.6%).

(Compound 65A)
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.54 (s, 1H, pNA-NH; D ₂ O exchange), 8.21 (d, 2H, J = 9.2 Hz, pNA C3,5-H), 8.20 (d, 1H, J = 8.2 Hz, NH; D ₂ O exchange), 8.11 (d, 1H, J = 8.2 Hz, NH; D ₂ O exchange), 7.83 (d, 2H, J = 9.2 Hz, pNA C2,6-H), 7.57 (d, 1H, J = 6.9 Hz, NH; D ₂ O exchange), 7.43-7.25 (m, 6H, benzyl-Ph, CONH ₂ a; D ₂ O exchange), 7.12 (s, 1H, CONH ₂ b; D ₂ O exchange), 5.31 (d, 1H, J = 6.9 Hz, MurNAc C4-OH; D ₂ O exchange), 4.74 (d, 1H, J = 3.4 Hz, MurNAc C1-βH), 4.67 (d, 1H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.61 (t, 1H, J = 6.0 Hz, MurNAc C6-OH; D ₂ O exchange), 4.44 (d, 1H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.35-4.17 (m, 3H), 3.87-3.77 (m, 1H), 3.74-3.60(m, 1H), 3.59-3.42 (m, 3H), 3.37-3.25 (m, 1H), 2.42 (t, 2H, J = 7.8 Hz, isoGln-βCH ₂ ), 2.13-1.99 (m, 1H, isoGln-γCH ₂ a), 1.89-1.74 (m, 1H, isoGln-γCH ₂ b), 1.79 (s, 3H, NHCO CH ₃ ), 1.26 (d, 3H, J = 7.1 Hz, D-Ala- CH ₃ or MurNAc- CH ₃ ), 1.24 (d, 3H, J = 7.6 Hz, Ala- CH ₃ or MurNAc- CH ₃ ).
LCMS m/z: 703.6 [M+H] ⁺ . (Exact Mass: 702.29)
Purity (LCMS): 100.00%

(Compound 65B)
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.53 (s, 1H, pNA-NH; D ₂ O exchange), 8.34 (d, 1H, J = 7.1 Hz, NH; D ₂ O exchange), 8.20 (d, 2H, J = 9.2 Hz, pNA C3,5-H), 8.10 (d, 1H, J = 8.0 Hz, NH; D ₂ O exchange), 7.82 (d, 2H, J = 9.2 Hz, pNA C2,6-H), 7.79 (d, 1H, J = 7.6 Hz, NH; D ₂ O exchange), 7.41-7.22 (m, 6H, benzyl-Ph, CONH ₂ a; D ₂ O exchange), 7.13 (s, 1H, CONH ₂ b; D ₂ O exchange), 5.39 (d, 1H, J = 6.9 Hz, MurNAc C4-OH; D ₂ O exchange), 4.83 (d, 1H, J = 3.4 Hz, MurNAc C1-βH), 4.66 (d, 1H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.61 (t, 1H, J = 5.9 Hz, MurNAc C6-OH; D ₂ O exchange), 4.51-4.31 (m, 3H), 4.29-4.19 (m, 1H), 3.76-3.59 (m, 2H), 3.58-3.42 (m, 3H), 3.40-3.28 (m, 1H), 2.44 (t, 2H, J = 8.0 Hz, isoGln-βCH ₂ ), 2.11-1.96 (m, 1H, isoGln-γCH ₂ a), 1.94-1.76 (m, 1H, isoGln-γCH ₂ b), 1.82 (s, 3H, NHCO CH ₃ ), 1.27 (d, 3H, J = 6.6 Hz, D-Ala- CH ₃ or MurNAc- CH ₃ ), 1.25 (d, 3H, J = 6.9 Hz, D-Ala- CH ₃ or MurNAc- CH ₃ )).
LCMS m/z: 703.6 [M+H] ⁺ . (Exact Mass: 702.29)
Purity (LCMS): 92.54%

<Deallylation (general method)>
The allyl ester (1 mmol) was dissolved in _CH3CN (75 mL) and water (3 mL), degassed under reduced pressure, and AcOH (5.0 eq.) and 4-methylmorpholine (4.0 eq.) were added. Pd(OAc) ₂ (0.5 eq.) and _PPh3 (1.0 eq.) were then added, and the mixture was stirred at 50°C for 6 hours. The disappearance of the raw materials was confirmed by TLC (10% MeOH/ _CHCl3 ), and the reaction solution was filtered (Millipore Milex-HV 0.45 μm) and concentrated to dryness under reduced pressure.

(Synthesis of benzyl-α-MurNAc-L-Ala-D-Glu(OH)-MCA (compound 66))

Benzyl-α-MurNAc-L-Ala-D-Glu(OAll)-MCA (compound 61) (100 mg, 0.13 mmol) was used in the reaction according to the general method, the suspension was removed by filtration, and the mixture was concentrated to dryness under reduced pressure. The residue was crystallized from n-Hex. (3 mL)/CHCl ₃ (3 mL), the supernatant was removed, and the residue was crystallized again from n-Hex. (3 mL)/CHCl ₃ (3 mL). The solid was filtered, washed with n-Hex./CHCl ₃ (1:1), and dried (60°C, reduced pressure) to obtain compound 66 (84 mg, 88.4%) as a pale yellow solid. ^1H -NMR and LCMS spectra suggested that the solid was a mixture of three isomers (abundance ratio approximately 3:1:1).
^1H -NMR (400 MHz, DMSO- _d6 ) δppm: 10.38, 10.36 (each s, 1H, coumarin C7-NH; _D2O exchange), 8.35-8.18 (br-m, 1H, NH; _D2O exchange), 7.87-7.67 (m, 3H, NH; _D2O exchange, coumarin C8-H [7.80 (d, J = 2.1 Hz), 7.78 (d, J = 2.1 Hz)], coumarin C5-H [7.73 (d, J = 8.7 Hz), 7.71 (d, J = 8.7 Hz)]), 7.59-7.47 (m, 1H, coumarin C6-H [7.56 (dd, J = 2.1, 8.7 Hz]), 7.41-7.19 (m, 5H _, O CH ₂ Ph), 6.27 (d, 1H, J = 1.1 Hz, Coumarin C3-H), 4.84-3.90 (m, 8H; C4-OH; D ₂ O exchange), 3.93 (br-t like, 0.65H, MurNAc C6-OH; D ₂ O exchange)]), 3.82-3.17 (m, 6H), 2.40 (d, 3H, J = 1,1 Hz, Coumarin C4- CH ₃ ), 2.32-2.20 (br-m, 2H, Glu-βCH ₂ ), 2.12-1.73 (m, 5H, Glu-γCH ₂ , NHCO CH ₃ [1.81, 1.80, 1.79 (each s, NHCO CH ₃ )]), 1.34-1.17 (m, 6H, Ala- CH ₃ and MurNAc- CH ₃ ).
LCMS m/z: 741.5 [M+H] ⁺ . (Exact Mass: 740.29)
Purity (LCMS; 326 nm): 91.63% (53.12%, 19.61%, 18.90%).

(Synthesis of benzyl-α-MurNAc-L-Ala-D-Glu (MCA)-OH (compound 67))

Benzyl-α-MurNAc-L-Ala-D-Glu(MCA)-OAll (compound 62) (200 mg, 0.26 mmol) was used in the reaction according to the general method, the suspension was removed by filtration, and the mixture was concentrated to dryness under reduced pressure. The residue was crystallized from n-Hex. (4 mL)/CHCl ₃ (4 mL), the supernatant was removed, and the residue was crystallized again from n-Hex. (4 mL)/CHCl ₃ (4 mL). The solid was filtered, washed with n-Hex., and dried (50°C, reduced pressure) to obtain compound 67 (189 mg, 89.5%) as a pale ochre solid. ^1H -NMR suggested that it was a mixture of two isomers (abundance ratio about 3:1).
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.52 (s, 0.75H, Coumarin C7-NH; D ₂ O exchange), 10.51 (s, 0.25H, Coumarin C7-NH; D ₂ O exchange), 8.41 (d, 0.25H, J = 7.6 Hz, NH; D ₂ O exchange), 8.26 (d, 0.25H, J = 7.6 Hz, NH; D ₂ O exchange), 8.16 (d, 0.75H, J = 7.6 Hz, NH; D ₂ O exchange), 8.12 (d, 0.75H, J = 8.0 Hz, NH; D ₂ O exchange), 7.76 (d, 0.25H, J = 2.1 Hz, coumarin C8-H), 7.75 (d-, 0.75H, J = 2.1 Hz, coumarin C8-H), 7.72 (d, 0.25H, J = 7.8 Hz, NH; D ₂ O exchange), 7.692 (d, 0.75H, J = 8.7 Hz, coumarin C5-H), 7.686 (d, 0.25H, J = 8.7 Hz, coumarin C5-H), 7.65 (d, 0.75H, J = 7.8 Hz, NH; D ₂ O exchange), 7.46 (dd, 0.75H, J = 2.1, 8.7 Hz, coumarin C6-H), 7.45 (dd, 0.25H, J = 2.1, 8.7 Hz, Coumarin C6-H), 7.40-7.23 (m, 5H, O CH ₂ Ph), 6.25 (d, 1H, J = 1.1 Hz; Coumarin C3-H), 4.81 (d, 0.25H, J = 3.4 Hz, MurNAc C1-βH), 4.75 (d, 0.75H, J = 3.4 Hz, MurNAc C1-βH), 4.66(d, 0.75H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.65(d, 0.25H, Jgem = 12.6 Hz, Ph- CH ₂ a), 4.52-4.26(m, 3H, [4.49(q, 0.25H, J = 6.6Hz), 4.43 (d, 0.75H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.42 (d, 0.25H, Jgem = 12.6 Hz, Ph- CH ₂ b), 4.32 (q, 0.75H, J = 6.6 Hz)]), 4.22-4.08 (m, 1H), 3.99-3.90 (br, 1H), 3.85-3.25 (m, 8H), 2.48-2.35 (m, 5H, Glu-βCH ₂ , [2.39 (d, 3H, J = 1.1 Hz, Coumarin C4- CH ₃ ]), 2.13-2.01 (m, 1H, Gln-γCH ₂ a), 1.95-1.75 (m, 1H, Gln-γCH ₂ b), 1.81 (s, 0.75H, NHCO CH ₃ ), 1.75 (s, 2.25H, NHCO CH ₃ ), 1.31-1.19 (m, 6H [1.26 (d, 2.25H, J = 6.6 Hz, Ala- CH ₃ or MurNAc- CH ₃ ), 1.24 (d, 2.25H, J = 6.9 Hz, Ala- CH ₃ or MurNAc- CH ₃ ]).
LCMS m/z: 741.6 [M+H] ⁺ . (Exact Mass: 740.29)
Purity (LCMS): 90.39%

<Acylation (acetylation, benzylation) (general method)>
HL-Ala-D-isoGln-pNA (compound 46) (1 mmol) was suspended in _CH2Cl2 (100 mL), _and TEA (1.05 eq.) and acid chloride (1.05 eq.) were added under ice cooling, and the mixture was stirred for 15 minutes, and then stirred at room temperature for 2.5 hours. MeOH (1 mL) was added, and the mixture was concentrated to dryness under reduced pressure.

(Synthesis of Ac-L-Ala-D-isoGln-pNA (compound 47))

H-Ala-D-isoGln-pNA (compound 46) (120 mg, 0.36 mmol) and acetyl chloride (29 mg, 0.37 mmol) were reacted according to the general method and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (5% to 10% MeOH-CHCl ₃ ). The fractions were combined, concentrated to dryness, and dried (50°C, reduced pressure) to give 122 mg of a white solid. This was suspended and washed with CHCl ₃ and dried (60°C, reduced pressure) to give compound 47 (69 mg, 51.1%). ^1H -NMR suggested that it was a mixture of two isomers (abundance ratio approximately 13:1).
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.56 (s, 1H, pNA-NH; D ₂ O exchange), 8.40 (d, 0.07H, J = 6.4 Hz, NH; D ₂ O exchange), 8.21 (d, 2H, J = 9.4 Hz, pNA C3,5-H), 8.164 (d, 1H, J = 8.2 Hz, NH; D ₂ O exchange), 8.155 (d, 0.93H, J = 6.4 Hz, NH; D ₂ O exchange), 7.83 (d, 2H, J = 9.4 Hz, pNA C2,6-H), 7.32 (s, 1H, isoGln- CONH ₂ a; D ₂ O exchange), 7.13 (s, 1H, isoGln- CONH ₂ b; D ₂ O exchange), 4.31-4.13 (m, 2H, L-Ala-αH and isoGln-αH), 2.42 (t, 2H, J = 7.8 Hz, D-isoGln-βCH ₂ ), 2.16-2.04 (m, 1H, isoGln-γCH ₂ a), 1.88-1.75 (m, 4H, CH ₃ CO [1.84 (s, 3H) and isoGln-γCH ₂ b), 1.23 (d, 0.21H, J = 7.1 Hz, Ala- CH ₃ ), 1.19 (d, 2.79H, J = 7.1 Hz, Ala- _CH3 ).
LCMS m/z: 380.5 [M+H] ⁺ . (Exact Mass: 379.15)
Purity (LCMS): 96.53% (88.33% + 8.20%)

(Synthesis of Benzoyl-L-Ala-D-isoGln-pNA (compound 48))

H-Ala-D-isoGln-pNA (compound 46) (120 mg, 0.36 mmol) and benzoyl chloride (52 mg, 0.37 mmol) were reacted according to the general method and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (5% to 10% MeOH-CHCl ₃ ). The fractions were combined, concentrated to dryness, and dried (50°C, reduced pressure) to obtain compound 48 (146 mg, 99.3%) as a white solid. ^1H -NMR suggested that it was a mixture of two isomers (abundance ratio approximately 13:1).
¹ H-NMR (400 MHz, DMSO-d ₆ ) δppm: 10.54 (s, 0.93H, pNA-NH; D ₂ O exchange), 10.53 (s, 0.07H, pNA-NH; D ₂ O exchange), 8.63 (d, 0.93H, J = 6.6 Hz, NH; D ₂ O exchange), 8.57 (d, 0.07H, J = 6.6 Hz, NH; D ₂ O exchange), 8.22 (d, 0.93H, J = 8.2 Hz, NH; D ₂ O exchange), 8.20 (d, 2H, J = 9.2 Hz, pNA C3,5-H), 7.98 (d, 0.07H, J = 8.2 Hz, NH; _D2O exchange), 7.92-7.86 (m, 2H, Bz C2,6-H), 7.82 (d, 2H, J = 9.2 Hz, pNA C2,6-H), 7.58-7.51 (m, 1H, Bz C4-H), 7.50-7.43 (m, 2H, Bz C3,5-H), 7.56 (s, 0.93H, isoGln- CONH ₂ a; D ₂ O exchange), 7.30 (s, 0.07H, isoGln- CONH ₂ a; D ₂ O exchange), 7.18 (s, 0.93H, isoGln- CONH ₂ b; D ₂ O exchange), 7.13 (s, 0.07H, isoGln- CONH ₂ b; D ₂ O exchange), 4.51-4.40 (m, 1H, Ala-αH), 4.30-4.16 (m, 1H, isoGln-αH), 2.44 (t, 2H, J = 7.8 Hz, isoGln-βCH ₂ ), 2.19-2.05 (m, 1H, isoGln-γCH ₂ a), 1.90-1.77 (m, 1H, isoGln-γCH ₂ b), 1.35 (d, 2.79H, J = 7.1 Hz, Ala- CH ₃ ), 1.25 (d, 0.21H, J = 7.1 Hz, Ala- CH ₃ ).
LCMS m/z: 442.5 [M+H] ⁺ . (Exact Mass: 441.16)
Purity (LCMS): 100.00%

Example 2: Evaluation of specificity of synthetic substrates
The synthesized substrate was reacted with Lc-Lys2 to evaluate the specificity of the enzyme reaction.

50 μL of 0.244 mmol/L synthetic substrate (DMF) was added to 145 μL of 20 mmol/L phosphate buffer (pH 6.0) and mixed well, then 5 μL of enzyme was added and incubated at 37°C for reaction. The specificity of the fluorescent substrate was measured using a multiplate reader (excitation 355 nm/emission 460 nm, measurement time 0.1 sec). The specificity of the chromogenic substrate was measured using a spectrophotometer (OD405).

(MCA substrate)
The MCA substrates, compounds 63, 67, 60 and 66, were used to react at 37°C for 4 hours, and the fluorescence was measured to evaluate the specificity. The measurement results of the specificity of each compound are shown in Table 1. The specificity of the synthetic substrates was expressed as the ratio of AMC produced in each reaction to the theoretical production amount of AMC (hydrolysis rate %).

Benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (compound 63) (63A: Rf = 0.25, 63B: Rf = 0.19; 10% MeOH/CHCl ₃ ), benzyl-α-MurNAc-L-Ala-D-Glu(MCA)-OH (compound 67) (isomer mixture ratio approx. 3:1), benzyl-α-MurNAc-L-Ala-D-Gln-MCA (compound 60) (isomer mixture ratio approx. 4:1) and benzyl-α-MurNAc-L-Ala-D-Glu(OH)-MCA 66 (isomer mixture ratio approx. The specificities (ratio of approximately 3:1:1) were 63A (19.1%), 63B (81.5%), 67 (5.2%), 60 (2.8%) and 66 (3.8%), respectively.

The D-Glu and D-Gln forms (compounds 67, 60, and 66) did not release AMC, indicating that they are not substrates for Lc-Lys2. On the other hand, the D-isoGln form (compound 63) was shown to be usable as a good substrate. Compound 63 exists as two presumed stereoisomers, 63A and 63B, and the highly polar 63B was shown to be a better substrate.

These results strongly suggest that the amino acid at the cleavage site of Lc-Lys2 is D-isoGln.

　The results of measuring the specificity when reacting with the MCA substrates 63, 45, 21, 30, 39 and 58 at 37°C for 60 minutes are shown in Tables 2 and 3.

The specificity of compound 63 was 63A (7.3%) and 63B (33.9%). Also, it was H-L-Ala-D-isoGln-MCA・HCl (compound 45) (20.0%), Fmoc-D-isoGln-MCA (compound 21) (23.4%), H-D-isoGln-MCA (compound 30) (1.3%), Fmoc-L-Ala-D-isoGln-MCA (compound 39) (7.6%), and benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-MCA (compound 58) (14.0%).

Compound 30 showed no specificity and was not shown to be a substrate. On the other hand, compounds 63, 45, 21, 39 and 58 showed specificity and were shown to be usable as substrates. These results indicate that compounds with an acyl group (Ala) or an acyloxy group (Fmoc group) of appropriate size in the amino group moiety can be used as substrates.

(pNA substrate)
Since it was suggested that the amino acid at the cleavage site of Lc-Lys2 is D-isoGln, benzyl-α-MurNAc-L-Ala-D-isoGln-pNA (compound 64) was synthesized in which 4NA was introduced into D-isoGln, and the specificity was also evaluated. The reaction was carried out using the pNA substrate at 37°C for 20 or 180 minutes, and the specificity was evaluated by measuring with a spectrophotometer. The measurement results of the specificity of each compound are shown in Table 4. The specificity of the synthetic substrate was expressed as the ratio of 4NA produced in each reaction to the theoretical production amount of 4NA (hydrolysis rate %).

Compound 64 existed in two presumed stereoisomers, 64A and 64B (64A: Rf = 0.12, 64B: Rf = 0.08; 10% MeOH/CHCl ₃ ). The specificity of these was 64A (43.0%, 97.0%) and 64B (97.4%, 97.3%). As with the MCA substrate, the highly polar 64B was shown to be a better substrate.

To investigate the effect of the optical isomer of Ala, benzyl-α-MurNAc-D-Ala-D-isoGln-pNA (compound 65) was synthesized and its specificity was examined. Compound 65 also had two presumed stereoisomers, 65A and 65B (65A: Rf = 0.14, 65B: Rf = 0.09; 10% MeOH/ CHCl ₃ ). The specificities of these were 65A (41.4%, 98.0%) and 65B (106.8%, 113.0%). This suggested that the stereoconfiguration of Ala had little effect on the specificity.

The specificity of the benzylidene-protected compound of compound 64, benzyl-4,6-O-benzylidene-α-MurNAc-L-Ala-D-isoGln-pNA (compound 56) (isomer mixture ratio approximately 17:3), was also evaluated. As a result, the specificity was 88.3% (20 minutes) and 106.8% (180 minutes), showing the same specificity as the debenzylidene derivative (compound 64B).

Furthermore, the specificity of the synthetic intermediates Fmoc-D-isoGln-pNA (compound 23), H-D-isoGln-pNA (compound 31), Fmoc-L-Ala-D-isoGln-pNA (compound 40) (isomer ratio approx. 9:1), H-L-Ala-D-isoGln-pNA (compound 46) (isomer ratio approx. 9:1), and the acyl derivative of compound 46, Ac-L-Ala-D-isoGln-pNA (compound 47) (isomer ratio approx. 13:1), and Bz-L-Ala-D-isoGln-pNA (compound 48) (isomer ratio approx. 13:1) were also examined. The reaction temperature and reaction time were 37°C and 30 min. The specificity measurement results are shown in Table 5.

Compound 31 showed no specificity, indicating that it cannot be used as a substrate. On the other hand, compounds 23 (69.8%), 40 (75.6%), 46 (67.0%), 47 (62.2%) and 48 (62.9%) showed moderate specificity, indicating that they can be used as substrates. These results indicate that compounds in which an acyl group or an acyloxy group (Fmoc group) of an appropriate size has been introduced into the amino group of compound 31 are likely to be substrates for Lc-Lys2.

(LCMS tracking results of compounds 63A and 63B)
The reaction of benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (compound 63A) (Rf = 0.25) and compound 63B (Rf = 0.19) with Lc-Lys2 was monitored by LCMS. The measurement results are shown in Table 6.

[Substrate]
A: Benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (Rf = 0.25) Compound 63A Exact Mass: 739.31
B: Benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (Rf = 0.19) Compound 63B Exact Mass: 739.31

[Product]
C: AMC Exact Mass: 175.06
D: Benzyl-α-MurNAc-L-Ala-D-isoGln-OH Exact Mass: 582.20

[Measurement sample]
a: Compound 63A reaction solution (substrate concentration: 61 μmol/L) and stock solution (0.244 mmol/L)
b: Compound 63B reaction solution (substrate concentration: 61 μmol/L) and stock solution (0.244 mmol/L)
c: AMC (0.244 mol/L)

[Measurement conditions]
Colume：Acquity UPLC BEH C18, 1.7μm, 2.1 X 50mm
Mobile phase: 0.1%HCO2H (A) / CH ₃ CN (B), Flow rate 0.7 mL/min., Gradient 0 min. (A: 91%) -3.2 min. (A: 19%) -3.7 min. (A: 19 > B: 91) -5 min. (B: 91)
Injection volume: 10 μL of reaction solution a and b, 0.5 μL of DMF solution of a (stock solution), b (stock solution), and AMC
Measurement wavelength: 326 nm (a and b stock solutions were measured at 270 nm)

In the LCMS measurement, the [M+H]+ of AMC and benzyl-α-MurNAc-L-Ala-D-isoGln-OH released by hydrolysis with Lc-Lys2 was observed ⁱⁿ the reaction solution of both compounds 63A and 63B. At the same time, unreacted compounds 63A and 63B were also confirmed. The specificity of these was compound 63A (19.0%) and compound 63B (95.6%), which was similar to the results of the measurement by fluorescence detection (Table 1).

These results indicate that benzyl-α-MurNAc-L-Ala-D-isoGln-MCA (63B) and benzyl-α-MurNAc-L-Ala-D-isoGln-pNA (64B) are particularly preferred substrates for use in this activity evaluation method.

The present invention has industrial applicability in that it provides a method for evaluating the activity of an enzyme for use in structural analysis of cell wall polysaccharides of lactic acid bacteria.

Claims (12)

A compound represented by the following formula (1) or a stereoisomer thereof:

...(1)
(In the above formula (1), A represents the following formula (2) or a protecting group for an amino group, and Z represents a detection group that becomes detectable when released.)

...(2)
(In the above formula (2), B represents a hydrogen atom, an N-acetyl-muramoyl group which may have a substituent, an acyl group, or a protecting group for an amino group.) The compound or stereoisomer thereof according to claim 1, wherein the protecting group for the amino group represented by B in the above formula (2) is an alkoxycarbonyl group, an alkenyloxycarbonyl group, or an aralkyloxycarbonyl group. The compound or stereoisomer according to claim 1, wherein the protecting group for the amino group represented by B in the above formula (2) is an Fmoc group (9-fluorenylmethyloxycarbonyl group), a Cbz group (benzyloxycarbonyl group), a Boc group (tert-butoxycarbonyl group) or an Alloc group (allyloxycarbonyl group). The compound or stereoisomer thereof according to claim 1, wherein the N-acetyl-muramoyl group, which may have a substituent and is represented by B in the above formula (2), is an N-acetyl-1-benzylmuramoyl group or an N-acetyl-1-benzyl-4,6-benzylidenemuramoyl group. The compound or stereoisomer thereof according to claim 1, wherein the acyl group represented by B in the above formula (2) is an acetyl group or a benzoyl group. The compound or stereoisomer thereof according to claim 1 or 2, wherein the protecting group for the amino group represented by A in the above formula (1) is an alkoxycarbonyl group, an alkenyloxycarbonyl group, or an aralkyloxycarbonyl group. The compound or stereoisomer thereof according to claim 1 or 2, wherein the protecting group for the amino group represented by A in the above formula (1) is an Fmoc group (9-fluorenylmethyloxycarbonyl group), a Cbz group (benzyloxycarbonyl group), a Boc group (tert-butoxycarbonyl group) or an Alloc group (allyloxycarbonyl group). 3. The compound or stereoisomer thereof according to claim 1 or 2, wherein A in the above formula (1) is the following formula (3), (4), (5), (6), (7), (8) or (9).

...(3)
(In the above formula (3), Ac represents an acetyl group, and Bn represents a benzyl group.)

...(4)
(In the above formula (4), Ac represents an acetyl group, Bn represents a benzyl group, and Ph represents a phenyl group.)

...(5)

...(6)

...(7)

...(8)

...(9) The compound or stereoisomer thereof according to claim 1 or 2, wherein Z in the above formula (1) represents a chromophore that emits color when released or a fluorescent group that emits fluorescence when released. 3. The compound or stereoisomer thereof according to claim 1 or 2, wherein Z in the above formula (1) is the following formula (10), (11), (12), (13), (14) or (15).

...(10)

...(11)

...(12)

...(13)

...(14)

...(15)
The compound or stereoisomer according to claim 1 or 2, which is used as a substrate for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase. A method for evaluating the activity of γ-D-glutamyl-L-lysyl endopeptidase, comprising the steps of:
A step of reacting γ-D-glutamyl-L-lysyl endopeptidase with a compound represented by the following formula (1) or a stereoisomer thereof;
and detecting released Z.

...(1)
(In the above formula (1), A represents the following formula (2) or a protecting group for an amino group, and Z represents a detection group that becomes detectable when released.)

... (2)
(In the above formula (2), B represents a hydrogen atom, an N-acetyl-muramoyl group which may have a substituent, an acyl group, or a protecting group for an amino group.)

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